WO2026022182A1

WO2026022182A1 - Detection of low allele frequency mutations using allele-specific amplification and crispr/cas13a-based method

Info

Publication number: WO2026022182A1
Application number: PCT/EP2025/071081
Authority: WO
Inventors: Sandrine DABERNAT; David Cappellen; Samuel AMINTAS
Original assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Centre Hospitalier Universitaire de Bordeaux; Universite de Bordeaux
Current assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Centre Hospitalier Universitaire de Bordeaux; Universite de Bordeaux
Priority date: 2024-07-23
Filing date: 2025-07-22
Publication date: 2026-01-29
Anticipated expiration: 2027-01-23

Abstract

To improve allele discrimination, the inventors adapted the system combining Cas13a detection sensitivity with allele-specific PCR amplification to propose CASPER (Cas13a Allele-Specific PCR Enzyme Recognition) as a new versatile, easy-to-implement, and highly sensitive method to detect low-frequency of sequence variant. CASPER enabled specific and sensitive detection of KRASG12D with low DNA input such as DNA extracted from patient's pancreatic ultrasound-guided fine-needle aspiration fluids. CASPER is easy to implement and a versatile reliable method virtually adaptable to any point mutation.

Description

DETECTION OF LOW ALLELE FREQUENCY MUTATIONS USING ALLELE¬

SPECIFIC AMPLIFICATION AND CRISPR/CAS13A-BASED METHOD

FIELD OF INVENTION

The present disclosure relates to medical diagnostics, specifically to methods for detecting genetic mutations associated with disease, in particular in pancreatic cancer.

BACKGROUND

Clustered Regulatory Insterspaced Short Palindromic Repeats (CRISPR)-Casl3a system has been shown very potent and sensitive for the detection exogenous sequences in human samples. For example, CRISPR-Casl3a coupled with fluorescent reporters was designed to detect specific RNA target sequences and was first applied to virus and bacteria genome detection with extremely high sensitivity (Gootenberg JS, et al. Science 2017 ; 356 : 438-442).

To establish its potential interest in oncology, CRISPR-Casl3a was used to detect cancerspecific large genomic alterations like EGFRVIII fusion variants or EGFR exon 19 deletions, with the performance required in clinical conditions (Cullot G, et al. CRISPR J. 2023 ; 6 : 140- 151). These large rearranged sequences can be considered exogenous since they are unique in the pathological genome and are absent in healthy genomes. CRISPR-Casl3a was originally also successful in distinguishing single nucleotide polymorphisms (allele frequency of 50%, (Gootenberg JS, et al. Science 2017 ; 356 : 438-442)).

However, point mutations occurring during oncogenesis can display very low allele frequencies, depending on the sample type, clonal frequency, or tumor heterogeneity. Therefore, there remains a need to develop new methods to detect such rare sequences in any challenging molecular situations, such as liquid biopsies or molecular residual disease follow-up in cancer patients.

This is the case for pancreatic ductal adenocarcinoma (PDAC) diagnosis. PDAC suffers from late diagnosis due to asymptomatic tumor growth and unspecific symptoms. Before any treatment is set, the carcinoma nature of the lesions is often confirmed by a cytopathological analysis. Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) providing tissue biopsies is risky (Storm AC, et al. World J. Gastroenterol. 2016 ; 22 : 8658-8669) and carries low negative predictive value delaying the diagnosis (Gobbi PG, et al. Cancer Epidemiol. 2013 ; 37 : 186-190). The inconclusive or doubtful results obtained by the EUS-FNA cytopathological exam are highly related to the scarcity of tumor cells in the samples.

The molecular diagnosis of PDAC has been developed by improving sensitivities of nucleic acid-based methods. KRASMUT alleles are very common in tumors and KRAS presents hotspots for mutations (Herdeis L, et al. Curr. Opin. Struct. Biol. 2021 ; 71 : 136-147). With this respect, PDAC is of particular interest since >90% of tumors present KRAS mutations (Bailey P, et al. Nature 2016 ; 531 : 47-52). The exploration of KRAS mutation status by PCR in the primary tumor coupled with cytology slightly improved the diagnosis performance (Bournet B, et al. J. Clin. Gastroenterol. 2015 ; 49 : 50-56; Cazacu IM, et al. Gastrointest. Endosc. 2021 ; 93 : 1142-1151. e2, Mansour Y, et al. Cancer Cytopathol. n.d. ; n/a) confirming the hypothesis that KRAS mutation detection participates in confirming the cancerous nature of the lesion, and expedites the therapeutic decision. Still, the intense desmoplastic reaction dilutes the informative tumor cells in fibrosis, highlighting the need for highly sensitive detection methods.

SUMMARY

The applicants challenged the detection of the 3 most frequent KRAS mutations (G12D, G12V, and G12C (Bryant KL, et al. Trends Biochem. Sci. 2014 ; 39 : 91-100) by CRISPR-Casl3a, using different crRNA guide designs, and after KRAS preamplification. To improve allele discrimination, the inventors adapted the system combining Casl3a detection sensitivity with allele-specific PCR amplification to propose CASPER (Casl3a Allele-Specific PCR Enzyme Recognition) as a new versatile, easy-to-implement, and highly sensitive method to detect low- frequency mutations. Indeed, CASPER enabled specific and sensitive detection of KRAS^G12D with low DNA input such as DNA extracted from patients’ pancreatic ultrasound-guided fine- needle aspiration fluids. CASPER is easy to implement and a versatile reliable method virtually adaptable to any point mutation.

The present disclosure relates to a method of detecting a sequence variant (such as a mutation) within a nucleic acid sample, preferably DNA sample, wherein said method comprises: a) selectively amplifying a target nucleic acid sequence comprising said variation (such as a mutation) from said sample by allele-specific polymerase amplification, preferably polymerase chain reaction amplification (PCR) or recombinase polymerase amplification (RPA), by contacting the sample to a pair of variant allele-specific primers and a DNA polymerase, preferably wherein a first AS primer comprises a sequence complementary to a part of the target nucleic acid sequence such that the 3 ’-end of the first primer binds to the sequence variant (e.g., mutation) in the nucleic acid target sequence, and more preferably further comprises a mismatch with the target nucleic acid sequence, preferably at the antepenultimate base of the first AS primer, b) in vitro transcribing amplified target nucleic acid sequence comprising said sequence variant (e.g., mutation) into target RNA, b) contacting said target RNA with a Cas protein having ribonuclease activity and a guide RNA comprising a complementary sequence to a part of the target RNA comprising said sequence variant (e.g., mutation), c) determining the ribonuclease activity, and d) optionally determining the allelic frequency of the sequence variant (e.g., mutation) in said sample.

In a more preferred embodiment, the second AS primer comprises a sequence complementary to a part of the target nucleic acid sequence and preferably further comprises a bacterial RNA polymerase promoter such as T7 promoter, T3 promoter or SP6 promoter and preferably the in vitro transcription of step b) is performed by adding a bacterial RNA polymerase, preferably selected from the group consisting of: T7 RNA polymerase, T3 RNA polymerase or SP6 polymerase.

In a particular embodiment, said Cas protein having ribonuclease activity is a Casl3a protein or functional variant thereof.

In a preferred embodiment, an RNA reporter, preferably comprising a fluorophore and a quencher is added in step c) and fluorescence intensity is measured in step d) to determine the ribonuclease activity. In a particular embodiment said sample is tumor DNA in biological fluids, more preferably comprising less than 20 ng of nucleic acid.

The present disclosure also relates to a method for identifying a subject having a mutation disease, said method comprising the method of detecting said mutation in a nucleic acid sample from said subject as described above, and wherein a higher ribonuclease activity or a higher mutation allelic frequency as compared to a corresponding control value is indicative that the subject has or is susceptible to have said disease, preferably said mutation disease is cancer, preferably selected from the group consisting of: pancreatic, colorectal, lung, ovarian and urogenital cancer, more preferably pancreatic cancer. In a preferred embodiment, said mutation is within an oncogene, preferably KRAS gene, more preferably wherein said mutation results in the mutation of a glycine at the position 12 or 13 of the KRAS protein, more preferably resulting in a KRAS G12D, G12V, G12C, G12A, G12S, G12R or G13D mutation.

In another aspect, the present disclosure relates to a therapeutic agent for use in the treatment of a mutation disease in a subject in need thereof, wherein a therapeutically efficient amount of a therapeutic agent is administered in a subject previously identified as having a mutation disease using a method as described above.

The present disclosure also relates to a method for evaluating a therapeutic response in a patient having a mutation disease, said method comprising the method of detecting a mutation in a nucleic acid sample as described above, wherein a decrease of the ribonuclease activity or the mutation allelic frequency during the treatment is indicative that the patient is responsive to the therapeutic agent, preferably said disease is a cancer, preferably selected from the group consisting of: pancreatic, colorectal, lung, ovarian and urogenital cancer, more preferably pancreatic cancer, and more preferably wherein said mutation is within an oncogene, preferably KRAS gene, more preferably wherein said mutation is KRAS G12 or G13 mutation, more preferably selected from the group consisting of: KRAS G12D, G12V, G12C, G12A, G12S, G12R and G13D mutation.

Finally, the present disclosure relates to a kit for detecting a sequence variant (e g., mutation) in a nucleic acid sample comprising: a) a first allele specific primer, preferably comprising a sequence complementary to a part of the target nucleic acid sequence such that the 3 ’-end of the primer binds to the sequence variant (e.g., mutation) in the nucleic acid target sequence, and optionally further comprises a mismatch with the target nucleic acid sequence, more preferably at the antepenultimate base of the first primer, b) a second primer comprising a sequence complementary to a part of the target nucleic acid and preferably further comprises a bacterial RNA polymerase promoter such as T7 promoter, T3 promoter or SP6 promoter, c) a guide RNA comprising a complementary sequence to a part of the target RNA comprising said sequence variant (e.g., mutation), and, optionally d) a Cas protein having ribonuclease activity, preferably Casl3a protein or a functional variant thereof.

In a more preferred embodiment, said kit is for detecting a mutation within KRAS gene resulting in KRASG12D mutation in a nucleic acid sample and comprises: a) a first AS primer comprising or consisting of a nucleic acid sequence of SEQ ID NO: 2, b) a second AS primer comprising or consisting of a nucleic acid sequence of SEQ ID NO: 3, preferably SEQ ID NO: 5, c) a guide RNA comprising or consisting of a nucleic acid sequence of SEQ ID NO: 7 and optionally, d) a Cas protein having ribonuclease activity, preferably Casl3a protein or a functional variant thereof.

FIGURE LEGENDS

Figure 1: crRNA19 for KRASG12X allele detection by CRISPR-Casl3a. (a-c)

Fluorescence ratio (left) and fluorescence level over time (right) in the presence of crRNA19G12D (a), crRNA19G12C (b) or crRNA19G12V (c) and PCR products from matching KRAS mutation or KRASWT/WT. (a-c) Results are presented as mean ± SEM with n=4 replicates from independent experiments.

Figure 2: crRNA19-14 for KRASG12X allele detection by CRISPR-Casl3a (a-c) Fluorescence ratio (left) and fluorescence level over time (right) in the presence of crRNA19G12D-14 G12D (a), crRNA19G12C-14 (b), or crRNA19G12V-14 (c) and PCR products from matching KRAS mutation or KRASWT/WT. (a-c) Results are presented as mean ± SEM with n=6 (a), n=4 (b) and n=2 (c) replicates from independent experiments.

Figure 3: crRNA12 for KRASG12D allele CASPER detection, (a-d) Fluorescence ratio (left) and fluorescence level over time (right) in the presence of crRNA12G12D (a), crRNA12G12D- 13 (b), crRNA12G12D-ll-13 (c), and PCR products from KRASG12D/G12D or KRASWT/WT. (d) Quantification of fluorescence ratio at 90 minutes in the presence of crRNA12G12D and PCR products from KRASG12D DNA diluted in KRASWT DNA. . (a-d) Results are presented as mean ± SEM with n=6 (a-b), n=4 (c) and n=3 (d) replicates from independent experiments. *: p<0.05; **: p<0.01; ns: non-significant.

Figure 4: Hairpin crRNA for KRASG12D allele CRISPR-Casl3a detection, (a-c) Fluorescence ratio (left) and fluorescence level overtime (right) in the presence of crRNAG12D hairpin 1 (b), crRNAG12D hairpin 2 (c) or crRNAG12D hairpin 3 (d) and PCR products from KRASG12D/G12D mutation or KRASWT/WT. (d) Quantification of fluorescence ratios at 90 minutes in presence of the crRNAG12D hairpin 3 and PCR products from KRASG12D DNA diluted in KRASWT DNA. (a-c) Results are presented as mean ± SEM with n=6 (b,d) and n=8 (c) replicates from independent experiments, (d) Results are presented as mean ± SEM with n=6 replicates from independent experiments. *: p<0.05; ***: p<0.001; ns : non-significant

Figure 5: Design of Allele-specific PCR primers and crRNA for CRISPR Casl3a-mediated KRASG12D detection, (a) The sequence of KRASG12D/G12D gDNA (SEQ ID NO: 1) was amplified using mutation-specific PCR primers (SEQ ID NO: 2 and 5). (b) Sequence of crRNA allele-specific (AS) G12D (SEQ ID NO: 7) and hybridization to T7 RNA products (SEQ ID NO: 6) from KRAS, (c) KRASG12D allelic frequency detected with AS qPCR depending on theoretical KRASG12D DNA dilutions, (d) KRASG12D allelic frequencies detected with ddPCR depending on theoretical KRASG12D DNA dilutions, (e) Four parameters of fit-curve analysis between sample KRASG12D theoretical allele frequency (0.01% to 100%) and fluorescence intensity ratio at 90 minutes in the presence of the crRNA ASG12D. (f) Spearman’s correlation analysis between sample KRASG12D theoretical allele frequency (0.01% to 10%) and fluorescence intensity ratio at 90 minutes in the presence of the crRNA ASG12D. (c-d) Results are presented as mean ± SD with n=4 (c) and n=6 (d) replicates from independent experiments. No positive droplets: Negative detection by digital droplet PCR.

Figure 6: Allele-specific PCR coupled with CRISPR-Casl3a for KRASG12D detection in patient’s pancreatic fine needle-aspiration samples, (a) Fluorescence ratio (left) and fluorescence level over time (right) in the presence of crRNA ASG12D and PCR products from KRASG12D/G12D or KRASWT/WT. (b) Quantification of fluorescence ratio at 90 minutes in the presence of the crRNA ASG12D and PCR products from KRASG12D DNA diluted in KRASWT DNA. (a-b) Results are presented as mean ± SEM with n=8 (a) and n=6 (b) replicates from independent experiments, (c) Experimental workflow for PDAC patient samples collection, sample processing and KRASG12D detection with ddPCR and CASPR. (d) Quantification of fluorescence ratio at 90 minutes of CAPSER assay for patients’ PDAC samples. Values at the top of the bars indicate mean fluorescence intensity ratios, doted lines indicate a fluorescence ratio of 1 (e) Quantification of fluorescence level over time with CASPER assay for blank, WT control, and positive patients’ samples, (d-e) The arrows point at the patient 6 results. Results are presented as mean ± SD with n=2 replicates from one experiment. *: p<0.05; **: p<0.01; ns: non-significant.

DETAILED DESCRIPTION

Definitions

As used herein, the term “nucleic acid sequence” refers to a single- or double-stranded nucleic acid. Said nucleic acid sequence can be DNA or RNA. In preferred embodiments, the “nucleic acid sequence” is a double-stranded DNA.

As used herein, the terms "complementary sequence" refers to the sequence part of a polynucleotide (e.g. part of crRNa) that can hybridize to another part of polynucleotides under standard low stringent conditions. Preferentially, the sequences are complementary to each other pursuant to the complementarity between two nucleic acid strands relying on Watson- Crick base pairing between the strands, i.e. the inherent base pairing between adenine and thymine (A-T) nucleotides and guanine and cytosine (G-C) nucleotides.

The term "subject" refers to both human and non-human animals. As used herein, the term “subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably, a subject according to the invention is a human.

The term "biological sample" or “sample” is generally obtained from a subject or from a population of subjects. A sample may be any biological tissue or fluid with which the sequence variant or mutation of the present disclosure may be identified. Frequently, a sample is a "clinical sample" (i.e., a sample obtained or derived from a patient to be tested). The sample may also be an archival sample with a known diagnosis, treatment, and/or outcome history. Examples of biological samples suitable for use in the practice of the present disclosure include, but are not limited to, bodily fluids, e.g., blood samples (e.g., blood smears), and cerebrospinal fluid, tumor tissue, or fine needle biopsy samples. Biological samples may also include sections of tissues, such as frozen sections taken for histological purposes. The term "biological sample" also encompasses any material derived by processing a biological sample. Derived materials include, but are not limited to, cells (or their progeny) isolated from the sample, as well as nucleic acid molecules (DNA and/or RNA) extracted from the sample. The biological sample may be tumor DNA present in biological fluids, such as circulating free DNA (cfDNA), which corresponds to degraded DNA fragments released to the blood plasma. cfDNA can be derived from healthy or tumor cells.

Method of detecting a sequence variant (e.g., mutation) in a nucleic acid sample

The inventors have developed a new method for the specific and sensitive detection of a sequence variant (e.g., mutation) with low nucleic acid sample input. The method increases the specificity of sequence variant (e.g., mutation) detection by selectively amplifying a target nucleic acid sequence containing the sequence variant from a sample using allele-specific polymerase amplification. This selective amplification ensures that subsequent steps in the method focus on the sequence of interest rather than the entire genomic content, resulting in more accurate and sensitive detection of mutations, even for point mutations or short rearrangement mutations and even when they are present at low allelic frequencies. The term “sequence variant” or “sequence variation” as used throughout the specification is intended to encompass any and all types of nucleic acid changes relative to another reference sequence.

The term “mutation” as used throughout the specification is intended to encompass any mutations and polymorphisms in the target nucleic acid molecule when compared to a wildtype allele of the same nucleic acid region. Such changes, include, but are not limited to deletions, duplications, insertions, translocations, inversions, genomic rearrangements, microsatellite instability, polymorphism, single nucleotide polymorphism and base substitutions of one or more nucleotides.

The method according to the present disclosure is sufficiently sensitive to detect point mutations or short rearrangement mutations. Therefore, in a preferred embodiment, the method according to the present disclosure allows to detect sequence variant of no more than 5, 4, 3, 2 or 1 nucleotide(s).

In another particular embodiment, the method according to the present disclosure can detect a sequence variant (e.g., mutation) present at low allelic frequencies, also named rare mutations.

The term “rare variation” or “rare mutation” as used herein and throughout the specification is intended to describe a sequence variant (e.g., mutation) in a nucleic acid molecule present in less than 40% of the nucleic acid molecules in the sample, preferably in less than 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.8%, 0.5%, 0.1%, 0.05%, 0.01%, or less compared to one or more, more common nucleic acid variants, which are referred to throughout the specification as the “wildtype” nucleic acid variants. In one embodiment, the rare variation (e g., mutation) is present in the sample in amount less than 10%, preferably less than 1%.

The method according to the present disclosure is sufficiently sensitive to detect a sequence variant (e.g., mutation) in low doses of tumor DNA, preferably in biological fluids, for example comprising less than 100 ng, preferably 90, 80, 70, 60, 50, 40, 30, even more preferably less than 20 ng of nucleic acid.

The sample may include one or more sequence variant (e.g., mutations) and there may also be one or more wildtype variants in the nucleic acid sample. The present disclosure relates to a method of detecting a sequence variant (e.g., mutation) in a nucleic acid (e.g. DNA) sample, wherein said method comprises: a) selectively amplifying a target nucleic acid sequence comprising said sequence variant (e.g., mutation) from said sample by allele-specific polymerase amplification, preferably polymerase chain reaction amplification (PCR) or recombinase polymerase amplification (RPA), by contacting said nucleic acid sample to a pair of variant allele-specific primers and a DNA polymerase, b) in vitro transcribing amplified target nucleic acid sequence comprising said sequence variant (e.g., mutation) into target RNA, c) contacting said target RNA with a Cas protein having ribonuclease activity and a guide RNA comprising a complementary sequence to a part of the target RNA sequence comprising said sequence variant (e.g., mutation), and d) determining the ribonuclease activity in said sample, and e) optionally determining the allelic frequency of the sequence variant (e.g., mutation) in said sample.

For selectively amplifying a target nucleic acid sequence comprising said sequence variant (e.g., mutation) from a nucleic acid (e.g. DNA) sample, the nucleic acid sample is subjected to an allele-specific (AS) polymerase amplification.

“Target nucleic acid sequence” or “target DNA sequence”, or a “target sequence” can be used interchangeably herein and relates to the fragment of the nucleic acid sample that is amplified by a pair of primers to form an amplicon. According to the disclosure, such target nucleic acid sequence includes the mutation to be identified (e g., includes deletion(s), addition(s) or substitution of at least one nucleotide, preferably no more than 5, 4, 3, 2 or 1 nucleotide(s)).

Allele-specific polymerase amplification method, also known as amplification refractory mutation system (ARMS) is a method used to amplify specifically an allele (allele of interest, for example sequence variant allele or mutated allele), distinct from another allele (non-targeted) by no more than 5 nucleotides. Allele-specific polymerase amplification exploits the fidelity of DNA polymerases which extend primers with mismatched 3’ base at much lower efficiency, from 100 to 100 000-fold less efficient than with a matched 3’ base (Chen, X., and Sullivan, P F, The Pharmacogeonomics Journal 2003; 3:77-96). The low efficiency in extending mismatched primers results in diminished polymerase amplification of the non-targeted allele.

To specifically amplify the allele of interest and not the non-targeted allele, the nucleic acid sample is subjected to allele of interest-specific primers (AS primers).

As used herein, a "primer pair" refers to a pair of oligonucleotide primers that each hybridizes to a specific target nucleotide sequence, in particular a forward primer that hybridizes to a first location of a nucleic acid sequence; and a reverse primer that hybridizes to a second location of the nucleic acid sequence downstream of the first location that anneal to opposite strands of a nucleic acid sequence so as to form an amplicon specific to the target sequence during the amplification reaction. Typically, the amplicon is produced by Polymerase chain reaction (PCR) or Recombinase polymerase amplification (RPA). The pair of primers can be designed using available computer programs. Preferably primers are designed to generate amplicons of at least 80 base pairs (bp) notably at least 90 bp, or at least 100 bp. In some embodiments, primers are designed to generate amplicons under 150 bp, for compatibility with circulating DNA detection.

Preferably, the primer pair is typically designed to have a melting temperature (Tm) lower than the critical denaturation temperature (Tc) of the reaction.

As used herein, the term “Tm'” or “melting temperature” of an oligonucleotide refers to the temperature (in degrees Celsius) at which 50% of the molecules in a population of a singlestranded oligonucleotide are hybridized to their complementary sequence and 50% of the molecules in the population are not-hybridized to said complementary sequence. The Tm of a primer or probe can be determined empirically by means of a melting curve. In some cases, it can also be calculated using formulas well known in the art (See, e.g., Maniatis, T, et al., Molecular cloning: a laboratory manual/Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y: 1982).

According to the present disclosure, the primer pair used in the present method is an allelespecific primer pair.

As used herein, the term “allele-specific primer”, “AS primer”, “variant allele-specific primer” refers to an oligonucleotide sequence that hybridizes to a sequence comprising an allele of interest (e.g. allele comprising a sequence variant (e.g., mutation)). Allele-specific primers are specific for a particular allele of a given target sequence (e.g., containing a sequence variant) and can be designed to detect a difference of as little as one nucleotide in the target sequence. In particular, the allele-specific primer comprises a nucleotide that can selectively hybridize and be extended from one target allele comprising sequence variant (e.g., mutation) at a given locus to the exclusion of the other (e.g. major or wild type allele) at the same locus.

The person skilled in the art would know how to design the allele-specific primer, for example by using the WASP web-based allele specific primer design tool as disclosed in Wwangkumhang P et al. BMC Genomics. 2007 Aug 14:8:275.

In particular, the variant allele-specific primer (e.g. forward AS primer) is designed to fully hybridize to the target sequence comprising said sequence variant (e g., mutation) so that the 3' end of the primer is at or near the site of a target sequence variant (e.g. mutation). In a particular embodiment, the nucleotide at the 3 'end of the primer (e.g. forward AS primer) directly overlies or binds to the sequence variant (e.g., mutation) in the target. This arrangement will maximize the chances that primer extension will only occur if there is a correct match between the nucleotide at the 3' end of the primer and the corresponding base in the target sequence. The primer will only extend if there is a 'match' between the nucleotide at the 3' end of the primer and the nucleotide at the site of the sequence variant (e.g., mutation) in the target sequence.

In a preferred embodiment, a first AS primer (e.g. forward primer) comprises a sequence complementary to a part of the target nucleic acid sequence such that the 3 ’-end of the first primer (e.g. forward primer) binds to the sequence variant (e.g., mutation) in the nucleic acid target sequence. This specific position ensures that the primer selectively binds to the target allele of interest and not the wild-type allele.

In a more preferred embodiment, to improve the specificity, a synthetic mismatch can also be added in the allele-specific primer (e.g. forward primer), preferably at the antepenultimate base of the variant allele-specific primer. This mismatch destabilizes the primer’s binding to nontarget alleles by at least 2 mismatches, enhancing specificity by preventing unintended amplification of non-target sequences. According to the present disclosure, the variant allele-specific primer is therefore designed to selectively amplify a target allele sequence comprising said sequence variant (e.g., mutation) by DNA polymerase and be refractory to the amplification of the wild-type allele.

"Amplifying", as used herein, refers to a process whereby multiple copies are made of one particular locus of a nucleic acid (i.e., a target sequence as mentioned above), such as genomic DNA. The nucleic acid amplification reaction may be any reaction in which a primer is extended by enzymatic addition of one or more nucleotides to it whilst that primer is bound or hybridized to a target sequence.

Such reactions include reactions that utilize thermal cycling such as the polymerase chain reaction (PCR) and ligase chain reaction (LCR) as well as isothermal amplification reactions such as nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), transcription-mediated amplification (TMA), Loop-Mediated Isothermal Amplification (LAMP) and rolling circle amplification, 3 SR, ramification amplification (as described by Zhang et al., Molecular Diagnosis (2001) 6 No 2, p 141-150), recombinase polymerase amplification (RPA) and others. In a preferred embodiment, amplification can be accomplished using PCR or RPA. In a preferred embodiment, polymerase amplification is a PCR amplification.

As used herein, the term “PCR amplifying” or “PCR amplification” refers generally to cycling polymerase-mediated exponential amplification of nucleic acids employing primers that hybridize to complementary strands, as described for example in Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press (1990). Devices have been developed that can perform thermal cycling reactions with compositions containing fluorescent indicators which are able to emit a light beam of a specified wavelength, read the intensity of the fluorescent dye, and display the intensity of fluorescence after each cycle. Devices comprising a thermal cycler, light beam emitter, and a fluorescent signal detector, have been described, e.g., in U.S. Pat. Nos. 5,928,907; 6,015,674; 6,174,670; and 6,814,934.

As used herein, the term “thermostable” or “thermostable polymerase” refers to an enzyme that is heat stable or heat resistant and catalyzes polymerization of deoxyribonucleotides to form primer extension products that are complementary to a nucleic acid strand. Thermostable DNA polymerases useful herein are not irreversibly inactivated when subjected to elevated temperatures for the time necessary to effect destabilization of single-stranded nucleic acids or denaturation of double- stranded nucleic acids during PCR amplification. Irreversible denaturation of the enzyme refers to substantial loss of enzyme activity. Preferably a thermostable DNA polymerase will not irreversibly denature at about 90°-100°C under conditions such as is typically required for PCR amplification.

In another particular embodiment, a recombinase polymerase amplification (RPA) reaction may be used to amplify the target nucleic acid sequence comprising said sequence variant (e.g., mutation) as described in Natoli M.E. et al. Anal Chem. 2021 Mar 23; 93(11): 4832-4840. RPA reactions employ recombinases which are capable of pairing sequence-specific primers with homologous sequence in duplex DNA, priming an isothermal DNA polymerase. If target nucleic acid sequence is present, nucleic acid amplification is initiated under isothermal conditions and no other sample manipulation such as thermal cycling or chemical melting is required. RPA reactions may be carried out at isothermal temperatures with an optimum reaction temperature of 37-42° C.

According to the present method, the amplified target nucleic acid sequence obtained as described above, is thereafter in vitro transcribed into target RNA.

The use of a bacterial RNA polymerase, for example selected from T7 RNA polymerase, T3 RNA polymerase, or SP6 polymerase, for the in vitro transcription step ensures a high yield of RNA transcripts from the amplified DNA. These RNA polymerases are highly specific to their respective promoters and are capable of rapid and robust transcription, which is critical for generating sufficient quantities of RNA for subsequent detection steps, especially when working with samples that have low nucleic acid concentrations.

In a particular embodiment, an RNA polymerase promoter, such as a T7 promoter, T3 RNA polymerase, or SP6 polymerase is added to one of the allele-specific primers, preferably the second allele-specific primer (e.g. reverse AS primer). In a preferred embodiment, the reverse allele-specific primer comprises a sequence complementary to a part of the target nucleic acid and further comprises, preferably at the 5’-end, a bacterial RNA polymerase promoter such as T7 promoter, T3 promoter or SP6 promoter. This results in an amplified nucleic acid (e.g. amplified DNA) comprising the target nucleic acid sequence containing said sequence variant (e g., mutation) and an RNA polymerase promoter. The in vitro transcription is therefore performed by adding a bacterial RNA polymerase, preferably selected from the group consisting of: T7 RNA polymerase, T3 RNA polymerase or SP6 polymerase. RNA polymerase will produce RNA from the double-stranded DNA templates. Preferably, the RNA polymerase can be added after or during the amplification reaction.

CRISPR system involves two components, Cas protein (CRISPR-associated protein) and guide RNA. Cas protein is a nuclease that uses guide RNA sequence as a guide to recognize and generate cleavage in RNA or DNA that is complementary to the guide RNA sequence.

According to the present disclosure, the CRISPR system used is RNA-guided RNases such as Type VI CRISPR-Cas system which targets RNA. Once activated by the binding to a target RNA sequence bearing complementary to the guide RNA (e.g. crRNA), Cas protein leads to RNA target cleavage and to "collateral" cleavage of non-targeted RNAs in proximity (Abudayyeh et al., 2016). This collateral RNA cleavage activity presents the opportunity to use RNA-guided RNases to detect the presence of a specific RNA by triggering in vitro nonspecific RNA degradation that can serve as a readout (Abudayyeh et al., 2016; East-Seletsky et al., 2016).

In a preferred embodiment, a Type VI CRISPR-Cas protein is a Cas protein having ribonuclease activity such as Casl3a, also named C2c2 or CasRx. The term “ribonuclease” refers to a wild type or variant enzyme capable of catalyzing the degradation ofRNAinto smaller components.

Typically, Cast 3a has two conserved HEPN domains (nucleotide-binding domains of higher eukaryotes and prokaryotes). These two domains are typically involved in the cutting of targeted mRNAs with complementary sequences for crRNA-guided recognition. Casl3a comprises two lobes termed the crRNA-recognition (REC) lobe and the nuclease (NUC) lobe. The Helical- 1 domain and the N-terminal domain (NTD) constitute the REC lobe, whereas the NUC lobe contains the HEPN1 domain, HEPN2 domain, Helical-2 domain, and a Linker between two HEPN domains.

By Cas protein having ribonuclease activity is also meant an engineered Cas protein such as Casl3a protein or a functional variant thereof which is capable of cleaving RNA target nucleic acid sequence and to "collateral" cleavage of non-targeted RNAs in proximity. Cas protein variant having ribonuclease activity may be a Cas protein that does not naturally exist in nature and that is obtained by protein engineering or by random mutagenesis. The Cas protein can be one type of the Cas proteins known in the art, homologs, orthologs thereof, or modified versions thereof. Preferably, Cas protein is Casl3a protein, homologs, orthologs thereof, or modified versions thereof.

According to a preferred embodiment, the Cas protein having ribonuclease activity is Casl3a protein or any functional variant thereof.

Cas 13a protein may be from an organism selected from the group consisting of: Leptotrichia, Listeria, Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma, and Campylobacter.

In certain embodiments, the Casl3a protein may be a Leptotrichia sp. Casl3p, preferably a Leptotrichia wadei (NCBI Reference Sequence: WP_314713413.1, updated on 11 Oct. 2023) or Leptotrichia buccalis Casl3a protein (NCBI Reference Sequence: WP 015770004.1, updated on 23-Sep 2020), more preferably Leptotrichia wadei Casl3a protein (Lwa Casl3a).

As used herein, the term "variant" or “functional variant” refers to a protein sequence that is derived from Casl3a protein as described above and comprises an alteration, i.e., a substitution, insertion, and/or deletion, at one or more positions, but retain the capacity of cleaving RNA target nucleic acid sequence and to "collateral" cleavage of non-targeted RNAs in proximity

The cleavage efficiency of a functional variant is similar to that of native casl3a protein in a cell when the ribonuclease activity measured for example with a RNA report comprising a quencher and fluorescent molecule with functional variants of Casl3a protein in a cell is similar than the control value (i.e., ribonuclease activity of native Casl3a protein), in particular the expression level varies by less than 40%, 30%, 20% or 10% of the control value.

The variant may be obtained by various techniques well known in the art. Examples of techniques for altering the nucleotide sequence encoding the native protein, include, but are not limited to, site-directed mutagenesis, random mutagenesis and synthetic oligonucleotide construction. As used herein, the term "variant" or “functional variant” may refer to a polypeptide having an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 98 or 99% sequence identity to a native Cast 3a protein as described above.

Cas protein having a ribonuclease activity is contacted with a guide RNA (gRNA) designed to comprise a complementary sequence to a part of the target RNA comprising said sequence variant (e.g., mutation) to specifically induce RNA cleavage within said target RNA and collateral cleavage of non-targeted RNAs in proximity.

As used herein, a “guide RNA”, “gRNA” or “single guide RNA” refers to a nucleic acid that promotes the specific targeting or homing of a gRNA/Cas complex to a target nucleic acid.

In particular, gRNA refers to an RNA that comprises a complementary sequence, also named crRNA, pairing with the target sequence recruits Cas having a ribonuclease activity to bind and target RNA. According to the present disclosure, crRNA is engineered to comprise a complementary sequence to a part of a target RNA comprising the sequence variant (e.g., mutation).

In a particular embodiment, the crRNA comprises a sequence of 5 to 50 nucleotides, preferably 15 to 30 nucleotides, which is complementary to a part of the RNA target comprising the sequence variant (e.g., mutation).

Said gRNA and/or Cas protein can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art and can be delivered to a cell using any- known techniques including but being not limited to calcium phosphate transfection, DEAE- Dextran transfection, electroporation, microinjection, biolistic, viral infection or liposome- mediated transfection. In another embodiment said gRNA and/or Cas protein are encoded by one or more nucleic acid constructs.

The term “nucleic acid construct” as used herein refers to a man-made nucleic acid molecule resulting from the use of recombinant DNA technology. A nucleic acid construct is a nucleic acid molecule, either single- or double-stranded, which has been modified to contain segments of nucleic acid sequences, which are combined and juxtaposed in a manner, which would not otherwise exist in nature. A nucleic acid construct usually is a “vector”, i.e. a nucleic acid molecule which is used to deliver exogenously created DNA into a host cell. The Cas protein having ribonuclease activity (e.g. Cas 13a) is activated by the hybridization of the guide RNA to the target RNA comprising said sequence variant (e.g., mutation) present in the sample. Once activated, said Cas protein having ribonuclease activity (e g. Cas 13a) non- specifically cleaves RNAs present in the sample.

The ribonuclease activity (i.e. cleavage of RNA) can be detected using any convenient detection methods (e.g., using an RNA reporter). This step provides a measurable output that correlates with the presence of the sequence variant (e.g., mutation), enabling the quantification of the sequence variant (e.g., mutation) and the determination of its allelic frequency within the nucleic acid sample.

In some cases, the present method includes a step of measuring a detectable signal produced by Cas protein-mediated RNA cleavage. The detectable signal can be any signal that is produced when RNA is cleaved. For example, in some cases the step of measuring can include one or more of: gold nanoparticle-based detection (e.g., see Xu et al., Angew Chem Int Ed Engl. 2007;46(19):3468-70; and Xia et. al., Proc Natl Acad Sci U S A. 2010 Jun 15;107(24): 10837- 41), fluorescence polarization, colloid phase transition/dispersion (e.g., Baksh et. al., Nature. 2004 Jan 8;427(6970): 139-41), electrochemical detection, semiconductor-based sensing (e.g., Rothberg et. al., Nature. 2011 Jul 20;475(7356):348-52; e.g., one could use a phosphatase to generate a pH change after RNA cleavage reactions, by opening 2' -3' cyclic phosphates, and by releasing inorganic phosphate into solution), and detection of a labeled detector RNA (see below for more details). The readout of such detection methods can be any convenient readout.

Examples of possible readouts include but are not limited to a measured amount of detectable fluorescent signal; a visual analysis of bands on a gel (e.g., bands that represent cleaved product versus uncleaved substrate), a visual or sensor-based detection of the presence or absence of a color (i.e., color detection method), and the presence or absence of (or a particular amount of) an electrical signal.

The use of a fluorescent reporter system in this context enhances the method's sensitivity, as it can detect low levels of ribonuclease activity that may correspond to rare sequence variant (e.g., mutation) within a sample. This is particularly advantageous when analyzing clinical samples with low nucleic acid concentrations or samples that contain a high background of wild-type sequences, as it can improve the detection of sequence variant (e g., mutation) that are present at low allelic frequencies. The ability to measure fluorescence intensity also facilitates the quantification of the allelic frequency of said sequence variant (e.g., mutation), providing valuable information for clinical diagnosis and treatment monitoring.

In a preferred embodiment, an RNA reporter, preferably a fluorescent RNA reporter, more preferably that includes a quencher/fluorophore pair is added with the CRISPR/Cas system to monitor the ribonuclease activity and then fluorescence intensity is measured to determine the ribonuclease activity level.

Upon target recognition and cleavage by the Cas-RNA complex, the fluorophore is separated from the quencher, leading to an increase in fluorescence intensity. This fluorescence signal provides a quantifiable measure of the ribonuclease activity, which correlates with the presence of the target sequence variant (e.g., mutation) in the sample. This approach allows for a sensitive and direct detection of the sequence variant (e g., mutation) without the need for additional labeling or detection steps, streamlining the process and potentially reducing the time to result.

According to the present disclosure, fluorescent molecule can be selected, for example, from the group consisting of FAM (5- or 6- carboxyfluorescein), VIC, NED, Fluorescein, FITC, IRD- 700/800, CY3, CY5, CY3.5, CY5.5, HEX, TET (5-tetrachloro-fluorescein), TAMRA, JOE, ROX, BODIPY TMR, Oregon Green, Rhodamine Green, Rhodamine Red, Texas Red, Yakima Yellow, Alexa Fluor PET, Biosearch Blue™, Marina Blue®, Bothell Blue®, Alexa Fluor®, 350 FAM™, SYBR® Green 1, Fluorescein, EvaGreen™, Alexa Fluor® 488 JOE™, 25 VIC™, HEX™, TET™, CAL Fluor®Gold 540, Yakima Yellow®, ROX™, CAL Fluor® Red 610, Cy3.5™, Texas Red®, Alexa Fluor® 568 Cry5™, Quasar™ 670, LightCycler Red640®, Alexa Fluor 633 Quasar™ 705, LightCycler Red705®, Alexa Fluor® 680, SYT0®9, LC Green®, LC Green® Plus+, and EvaGreen™. Preferably, the detectable label is selected from 6- carboxyfluorescein, FAM, or tetrachlorofluorescein, (acronym: TET), Texas Red, Cyanin 5, Cyanine 3, or VIC™. Selection of adapted fluorophores is classical in the field can be typically achieved according to general recommendations. As a matter of example, well-suited fluorophores include FAM and VIC as used in the experimental results.

Typical quenchers are tetramethylrhodamine, TAMRA, Black Hole Quencher or nonfluorescent quencher. The quencher molecule quenches the fluorescence emitted by the fluorophore when excited by the cycler’s light source typically via FRET (Forster Resonance Energy Transfer). As long as the fluorophore and the quencher are in proximity, quenching inhibits any fluorescence signals.

In a similar way, fluorescence energy transfer (FRET) may be used to generate a detectable positive signal in the RNA report. Thus, in the context of the embodiments disclosed herein, the fluorophore/quencher pair is replaced with a donor fluorophore/acceptor pair attached to the RNA reporter. When intact, the RNA reporter generates a first signal (negative detectable signal) as detected by the fluorescence or heat emitted from the acceptor. Upon activation of the Cas protein, the RNA reporter is cleaved and FRET is disrupted such that fluorescence of the donor fluorophore is now detected (positive detectable signal).

The fluorescence signal associated with the ribonuclease activity can be measured by any method known in the art, for example with optical detector such as the CFX96 Touch Real- Time PCR Detection System (Bio-Rad) and the fluorescence level can be quantified at different time points. Results analysis can be performed using CFX MaestroTM software (BioRad). A threshold, under which a fluorescent signal is considered as “a residual fluorescent signal” can be determined by the one skilled in the art according to classical signal analysis techniques. Said threshold can be typically the fluorescence intensity in a control sample, for example from a sample comprising only wild-type target nucleic acid sequence. The fluorescent intensity in the sample can therefore be normalized to fluorescent intensity of control sample (e.g. comprising wild-type target nucleic acid sequence)

In a particular embodiment, the fluorescence intensity ratio may be calculated as follows:

((variant template fluorescence intensity at the end-point - variant template fluorescence intensity at the initial time point)) / ((Wild-type template fluorescence intensity at end-point - Wild-type template fluorescence intensity at the initial time point)).

The inventors in the present application showed that the fluorescence intensity, in particular fluorescence intensity as measured above is fully quantitative and the allelic frequency of the sequence variant (e.g., mutation) can be thereafter deduced from the fluorescence intensity ratio, for example with a pre-established calibration curve. In a preferred embodiment, the method according to the present disclosure is particularly suitable to detect low-frequency alleles, in particular in a sample comprising low concentration of DNA (e.g., comprising less than 20 ng of nucleic acid).

This method is therefore particularly suitable for detecting a sequence variant (e g., mutation) within an oncogene from a subject’s nucleic acid sample (e g. DNA sample) that may have a very low allele frequency and/or contain a low nucleic acid concentration, such as in liquid biopsies.

An oncogene is a gene that has the potential to cause cancer. There are several categories of oncogenes commonly used. As non-limiting examples categories of oncogenes can be selected from the group consisting of: grow factors or mitogens oncogenes such as C-Sis involved for example in glioblastomas, fibrosarcoma, osteosarcomas, breast carcinomas and melanomas, Receptor tyrosine kinases oncogenes such as epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR) and vascular endothelial growth factor receptor (VEGFR), HER2/neu involved for example in breast cancer, gastrointestinal stromal tumors, non-small-cell lung cancer and pancreatic cancers, cytoplasmic tyrosine kinase oncogenes such as Src-family, Syk-ZAP-70 family, BTK family of tyrosine kinase, and Abl gene involved for example in colorectal and breast cancers, melanomas, ovarian cancers, gastric cancers, head and neck cancers, pancreatic cancer, lung cancer, brain cancers, and blood cancers, Cytoplasmic Serine/threonine kinases and their regulatory subunits oncogenes such as Raf kinase, and cyclin-dependent kinases involved for example in malignant melanoma, papillary thyroid cancer, colorectal cancer, and ovarian cancer, regulatory GTPases such as Ras gene or KRAS gene involved for example in adenocarcinomas of the pancreas and colon cancers, thyroid tumors, and myeloid leukemia, transcription factors oncogenes such as myc gene involved for example in malignant T-cell lymphomas and acute myeloid leukemias, breast cancer, pancreatic cancer, retinoblastoma, and small cell lung cancer and Transcriptional coactivators oncogenes such as YAP and WWTR1 genes, involved for example in glioma, melanoma, lung cancer, and breast cancers.

In a particular embodiment, the method according to the present disclosure is a method of detecting a sequence variant (e.g., mutation) within an oncogene in a subject nucleic acid (e g. DNA) sample, wherein said method comprises: a) selectively amplifying a target nucleic acid sequence comprising said sequence variant (e.g., mutation), from a nucleic acid sample by allele-specific polymerase amplification, (e.g., PCR or RPA) by contacting the nucleic acid sample to a pair of variant allele-specific primers and a DNA polymerase, b) in vitro transcribing amplified nucleic acid sequence comprising said sequence variant (e.g., mutation) into target RNA, c) contacting said target RNA with a Cas protein having ribonuclease activity (e.g. Casl3a or a functional variant thereof), a guide RNA comprising a complementary sequence to a part of the target RNA sequence comprising said sequence variant (e g., mutation), and preferably an RNA reporter, more preferably comprising a fluorophore and a quencher, d) determining the ribonuclease activity, and e) optionally determining the allelic frequency of sequence variant (e.g. mutation) in said sample.

In a more particular embodiment, the method according to the present disclosure is a method of detecting a sequence variant (e.g., mutation) within an oncogene in a subject nucleic acid (e g. DNA) sample, wherein said method comprises: a) selectively amplifying a target nucleic acid sequence comprising said sequence variant (e.g., mutation), from a nucleic acid sample by allele-specific polymerase amplification (e.g., PCR or RPA), by contacting the nucleic acid sample to a pair of variant allele-specific (AS) primers and a DNA polymerase, wherein a first AS primer comprises a sequence complementary to a part of the target nucleic acid sequence such that the 3 ’-end of the primer binds to the sequence variant (e.g., mutation) of the nucleic acid target sequence, and preferably further comprises a mismatch with the target nucleic acid sequence (e g. at the antepenultimate base), and, wherein a second AS primer comprises a sequence complementary to a part of the target nucleic acid sequence, and preferably further comprises a bacterial RNA polymerase promoter such as T7 promoter, T3 promoter or SP6 promoter, b) in vitro transcribing amplified nucleic acid sequence comprising said sequence variant (e.g., mutation) into target RNA, preferably by adding a bacterial RNA polymerase, preferably selected from the group consisting of: T7 RNA polymerase, T3 RNA polymerase or SP6 polymerase, c) contacting said target RNA with a Cas protein having ribonuclease activity (e.g. Casl3a or a functional variant thereof), a guide RNA comprising a complementary sequence to a part of the target RNA comprising said sequence variant (e.g., mutation), and preferably an RNA reporter, more preferably comprising a fluorophore and a quencher, d) determining the ribonuclease activity, and e) optionally determining the allelic frequency of the sequence variant (e.g., mutation) in said sample.

In a more preferred embodiment, the step b) and c) are performed simultaneously.

In a preferred embodiment, the method according to the present disclosure allows to detect a sequence variant (e.g., mutation) within an oncogene such as KRAS gene (Gene ID: 3845, updated on 16-May-2024). In a more preferred embodiment, the method according to the present disclosure allows to detect a mutation within the KRAS gene resulting in the mutation of a glycine at the position 12 or 13 of the KRAS protein (UniprotKB: P01116 • RASK_HUMAN, updated on 27-March-2024), more preferably resulting in a KRAS G12D, G12V, G12C, G12A, G12S, G12R or G13D mutation, preferably KRAS G12D, G12V, or G12C mutation.

In the particular embodiment described in the paragraph above, the genomic target sequence encoding KRAS^G12D mutation, allele specific forward and reverse primers and RNA target sequence obtained after allele-specific amplification and in vitro transcription and guide RNA used are represented in the Table 1 below.

Table 1 : Genomic target sequence comprising KRAS gene with a mutation (bold) (SEQ ID NO: 1) resulting in KRAS³¹²⁰ protein mutation amplified with forward and reverse allelic specific (AS) primers (SEQ ID NO: 2 and 5, respectively). In forward AS primer, the mismatch is underlined and in grey and mutation is in bold. In reverse AS primer, the sequence encoding T7 promoter (SEQ ID NO: 4) is underlined and the sequence complementary to genomic target sequence is in bold (SEQ ID NO: 3). RNA target sequence (SEQ ID NO: 6) is obtained after AS polymerase amplification and in vitro transcription. Within RNA target sequence, the complementary sequence to crRNA (SEQ ID NO: 7) is underlined, the mismatch is indicated in bold and grey and the mutation in bold. In a particular embodiment, the method according to the present disclosure is a method of detecting a mutation within KRAS gene in a subject nucleic acid (e g. DNA) sample, wherein said method comprises: a) selectively amplifying a target nucleic acid sequence comprising said mutation from said sample by allele-specific polymerase amplification (e.g., PCR or RPA), by contacting the nucleic acid sample to a pair of variant allele-specific primers and a DNA polymerase, b) in vitro transcribing amplified nucleic acid sequence comprising said mutation into a target RNA, f) contacting said target RNA with a Cas protein having ribonuclease activity (e.g. Casl3a protein and a functional variant thereof), a guide RNA comprising a complementary sequence to a part of the target RNA comprising said mutation, and preferably an RNA reporter, more preferably more preferably comprising a fluorophore and a quencher, c) determining the ribonuclease activity, and d) optionally determining the mutation allelic frequency in said sample.

In a more particular embodiment, the method according to the present disclosure is a method of detecting a mutation within KRAS gene in a subject nucleic acid (e.g. DNA) sample, wherein said method comprises: a) selectively amplifying a target nucleic acid sequence comprising said mutation from said sample by allele-specific polymerase amplification (e.g., PCR or RPA), by contacting the sample to a pair of variant allele-specific primers and a DNA polymerase, wherein a first AS primer comprises a sequence complementary to a part of the target nucleic acid sequence such that the 3 ’-end of the primer binds to the mutation of the nucleic acid target sequence, and preferably further comprises a mismatch with the target nucleic acid sequence (e.g. at the antepenultimate base) and, wherein a second AS primer comprises a sequence complementary to a part of the target nucleic acid sequence and preferably further comprises a bacterial RNA polymerase promoter such as T7 promoter, T3 promoter or SP6 promoter, b) in vitro transcribing amplified nucleic acid sequence comprising said mutation into target RNA, preferably by adding a bacterial RNA polymerase, more preferably selected from the group consisting of: T7 RNA polymerase, T3 RNA polymerase or SP6 polymerase c) contacting said target RNA with a Cas protein having ribonuclease activity (e.g., Casl3a protein or a functional variant thereof), a guide RNA comprising a complementary sequence to a part of the target RNA comprising said mutation, and preferably an RNA reporter, more preferably more preferably comprising a fluorophore and a quencher, d) determining the ribonuclease activity, and e) optionally determining the mutation allelic frequency in said sample.

In a more particular embodiment, the present disclosure relates to a method of detecting a mutation within the KRAS gene resulting in the mutation of a glycine at the position 12 or 13 of the KRAS protein, more preferably resulting in a KRAS G12D, G12V or G12C mutation in a subject sample

In another particular embodiment, the method according to the present disclosure is a method of detecting KRAS^G12D mutation in a subject nucleic acid sample, wherein said method comprises: a) selectively amplifying a target nucleic acid sequence comprising a mutation within KRAS gene resulting in KRAS^G12D mutation from a nucleic acid sample by allele-specific polymerase amplification, (e.g., PCR or RPA) by contacting the nucleic acid sample to a pair of variant allele-specific primers and a DNA polymerase, b) in vitro transcribing amplified nucleic acid sequence comprising said mutation into target RNA, c) contacting said target RNA with a Cas protein having ribonuclease activity (e.g. Casl3a protein or a functional variant thereof) and a guide RNA comprising a complementary sequence to a part of the target RNA comprising said mutation, d) determining the ribonuclease activity, and e) optionally determining the mutation allelic frequency in said sample.

In a more particular embodiment, the method according to the present disclosure is a method of detecting KRAS^G12D mutation in a subject nucleic acid sample, wherein said method comprises: a) selectively amplifying a target nucleic acid sequence comprising a mutation within KRAS gene resulting in KRAS^G12D mutation, preferably comprising or consisting of SEQ ID NO: 1, from a nucleic acid sample by allele-specific polymerase amplification (e.g., PCR or RPA), by contacting the nucleic acid sample to a pair of variant allele-specific primers and a DNA polymerase, wherein a first AS primer comprises a sequence complementary to a part of the target nucleic acid sequence such that the 3 ’-end of the primer binds to the mutation of the nucleic acid target sequence, and preferably further comprises a mismatch with the target nucleic acid sequence (e.g. at the antepenultimate base), more preferably comprising or consisting of sequence of SEQ ID NO: 2 and wherein a second AS primer comprises a sequence complementary to a part of the target nucleic acid sequence, preferably the second AS primer comprising of consisting of SEQ ID NO: 3, and more preferably further comprises a bacterial RNA polymerase promoter such as T7 promoter, T3 promoter or SP6 promoter, again more preferably the second AS primer comprising of consisting of SEQ ID NO: 5, b) in vitro transcribing amplified nucleic acid sequence comprising said mutation into target RNA, preferably by adding a bacterial RNA polymerase, preferably selected from the group consisting of: T7 RNA polymerase, T3 RNA polymerase and SP6 polymerase c) contacting said target RNA with a Cas protein having ribonuclease activity (e.g. Cast 3a and a functional variant thereof), a guide RNA comprising a complementary sequence to a part of the target RNA comprising said mutation, preferably a guide RNA comprising or consisting of SEQ ID NO: 7, and preferably a RNA reporter, more preferably more preferably comprising a fluorophore and a quencher, d) determining the ribonuclease activity, and e) optionally determining the mutation allelic frequency in said sample.

In some embodiments, the method comprises a single gRNA and Cast 3a protein capable of detecting a single sequence variant (e.g., mutation). In another particular embodiment, said method may comprise at least two different gRNAs capable of detecting several sequence variants (e.g., mutations) within the DNA sample. In particular, said gRNA and Cas protein can be used successively in such a way that a gRNA/Casl3a protein cleaves a first target sequence comprising a first sequence variant (e.g., mutation). Once first sequence variant (e.g., mutation) event is detected, a second gRNA/Cas may be used to cleave a second target sequence comprising a second sequence variant (e.g., mutation) event within said or another target sequence.

A method for identifying a subject having a mutation disease.

The detection of a mutation in a nucleic acid subject sample can be indicative that a subject has or is susceptible to have a mutation disease (i.e., a disease associated with the mutation), such as a genetic disease. On the contrary, if no mutation is detected in said nucleic acid subject sample, this is indicative that said subject has not or is not susceptible to have a mutation disease.

Therefore, the detection of a mutation according to the method as mentioned above can be used in the diagnosis of a mutation disease, such as genetic disease, in particular genetic cancers or cancer predisposition.

Activation of ribonuclease activity in said subject sample as determined by the method described above compared to a negative and a positive control value is indicative that the subject has or is susceptible to have a mutation disease (e.g. genetic disease such as cancer).

The present disclosure also relates to a method for identifying a subject having a mutation disease (e.g. genetic disease such as cancer), comprising detecting a mutation in a nucleic acid (e g. DNA) subject sample according to the method as described above, in particular by: a) selectively amplifying a target nucleic acid sequence comprising said mutation from said sample by allele-specific polymerase amplification (e.g. PCR or RPA), by contacting said nucleic acid sample to a pair of variant allele-specific primers as described above and a DNA polymerase, b) in vitro transcribing amplified target nucleic acid sequence comprising said mutation into target RNA, c) contacting said target RNA with a Cas protein having ribonuclease activity (e.g. Casl3a protein of a functional variant thereof), a guide RNA comprising a complementary sequence to a part of the target RNA sequence comprising said mutation, and preferably an RNA report, and d) determining the ribonuclease activity, and optionally determining the mutation allelic frequency in said sample, wherein a higher ribonuclease activity or higher mutation allelic frequency in said subject sample compared to a control value is indicative that the subject has or is susceptible to have a mutation disease.

Typically, a nucleic acid (e.g. DNA) sample according to the invention is obtained from a patient, preferably a cancer patient. The terms "subject" and "patient" are used interchangeably herein and refer to both human and non-human animals. As used herein, the term “patient” or “subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably, a patient or a subject according to the invention is a human.

In a preferred embodiment, the method for identifying a subject having a mutation disease further comprises a step of comparing the ribonuclease activity or the mutation allele frequency in said subject sample determined by the method as described above to a corresponding control value.

According to the present disclosure, the term “threshold value”, “control value” or “cut-off value” can be used interchangeably and can be determined experimentally, empirically, or theoretically. A threshold value refers to the ribonuclease activity or the mutation allele frequency in a biological sample obtained from a general population or from a selected population of subjects. For example, the general population may comprise apparently healthy subjects, such as individuals who have not previously had any signs or symptoms indicating the presence of said mutation disease. The term "healthy subjects" as used herein refers to a population of subjects who do not suffer from any known condition, and in particular, who are not affected by said mutation disease. In another embodiment, the threshold value refers to the ribonuclease activity or the mutation allele frequency in a biological sample obtained from patients who is not diagnosed with a mutation disease. Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the ribonuclease activity or the mutation allele frequency in a group of reference, one can use algorithmic analysis for the statistical treatment of the measured values in samples to be tested, and thus obtain a classification standard having significance for sample classification. In a particular embodiment, Receiver operating characteristic (ROC) analysis was performed to calculate the ribonuclease activity or the mutation allele frequency cut-off value of using DNA samples with known mutation status. The ribonuclease activity or the mutation allele frequency offering the highest sensitivity and specificity were selected as cut-off points as described in the examples of the present application. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc. According to the present disclosure, the threshold value can be determined for each sequence variant (e.g., mutation) evaluated.

A mutation disease according to the present disclosure is a disease caused by a change in the DNA sequence, in particular a disease caused by a mutation in at least one gene (i.e., genetic disease).

A genetic disease is a disease caused in whole or in part by a change in the DNA sequence away from the normal sequence. Genetic disease can be caused by a mutation in one gene, in multiple genes, a combination of gene mutations and environmental factors or damage to chromosomes.

As non-limiting examples, genetic diseases can be selected from the group consisting of: achondroplasia caused by a gene alteration in FGFR3 gene, alpha- 1 antitrypsin deficiency, autism, autosomal dominant polycystic kidney disease, breast cancer, - Colon cancer, Chron’s Disease, Cystic Fibrosis, Dercum Disease, Duane syndrome, Duchene Muscular dystrophy, Factor V Leiden Thrombophilia, Familia hypercholesterolemia, familial mediterranean fever, Fragile X syndrome, Gaucher disease, Hemochromatosis, hemophilia, Holoprosencephaly_? inborn errors of metabolism, Marfan syndrome, Methylmalmonic Acidemia, Myotonic Dystrophy, Neurofibromatosis, Noonan Syndrome, Osteogenesis Imperfecta, Parkinson’s disease, Phenylketonuria, Poland anomaly, Porphyria, Progeria, Prostate cancer, Retinitis Pigmentosa, Severe combined Immunodeficiency, Sickle cell disease, Skin cancer, Spinal muscular atrophy, Tay-Sachs Disease, Thalassemia, Trimethylaminuria, Velocardiofacial syndrome, pancreatic cancer and Wilson disease, preferably pancreatic, colorectal, lung, ovarian and urogenital cancer, more preferably pancreatic cancer , more preferably pancreatic ductal adenocarcinoma. In a preferred embodiment, the genetic disease is a cancer, preferably selected from the group consisting of: adenoma or primary tumors, such as colorectal cancer (also called colon cancer or large bowel cancer), colon adenocarcinoma, rectal adenocarcinoma, gastric cancer, stomach cancer, endometrial cancer, uterine cancer, uterine corpus endometrial carcinoma, breast cancer, bladder cancer, hepatobiliary tract cancer, liver hepatocellular carcinoma, urinary tract cancer, urothelial carcinoma, ovary cancer, ovarian serous cystadenocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma, bladder cancer, prostate cancer, kidney cancer, kidney renal papillary cell carcinoma, head and neck cancer, skin cancer, skin cutaneous melanoma, thyroid carcinoma, squamous cell carcinoma, lymphomas, leukemia, brain cancer, brain lower grade glioma, glioblastoma, glioblastoma multiforme, astrocytoma, and neuroblastoma, preferably pancreatic cancer , more preferably pancreatic ductal adenocarcinoma.

In a particular embodiment, the detection of a mutation within KRAS gene in a subject sample using a method as described above is indicative that said subject has or is susceptible to have a cancer, preferably a pancreatic cancer such as pancreatic ductal adenocarcinoma (PDAC).

In a more particular embodiment, the detection of a mutation within the KRAS gene resulting in the mutation of a glycine at the position 12 or 13 of the KRAS protein, more preferably resulting in a KRAS G12D, G12V, G12C, G12A, G12S, G12R orG13D mutation, again more preferably KRAS G12D, G12V or G12C mutation in a subject sample using a method as described above is indicative that said subject has or is susceptible to have a pancreatic cancer such as pancreatic ductal adenocarcinoma (PDAC).

In a more particular embodiment, the detection of a mutation within the KRAS gene resulting in a KRAS G12D mutation in a subject sample using a method as described above is indicative that said subject has or is susceptible to have a pancreatic cancer such as pancreatic ductal adenocarcinoma (PDAC).

In another particular embodiment, the present disclosure relates to the therapeutic use of a therapeutic agent in a patient in need thereof wherein said therapeutic agent is administered to said patient who is previously identified as having a mutation disease by a method as described above. In a particular embodiment, the present disclosure relates to a method of treating a mutation disease, in particular genetic disease such as cancer in a patient in need thereof, comprising detecting a mutation in a nucleic acid (e.g. DNA) sample previously collected from said patient according to the method as described above, in particular by: a) selectively amplifying a target nucleic acid sequence comprising said mutation from said sample by allele-specific polymerase amplification (e.g. PCR or RPA), by contacting said nucleic acid sample to a pair of variant allele-specific primers as described above and a DNA polymerase, b) in vitro transcribing amplified nucleic acid sequence comprising said mutation into target RNA, c) contacting said target RNA with a Cas protein having ribonuclease activity (e.g. Casl3a or a functional variant thereof), a guide RNA comprising a complementary sequence to a part of the target RNA comprising said mutation, and preferably an RNA reporter, d) determining the ribonuclease activity, and optionally determining the mutation allele frequency in said sample, wherein a higher ribonuclease activity or higher mutation allelic frequency in said subject sample compared to a control value is indicative that the subject has a mutation disease, and e) administering a therapeutically efficient amount of a therapeutic agent (e.g. chemotherapy, immunotherapy, targeted agent) in said patient identified as having a mutation disease.

The person skilled in the art would know to adapt the treatment according to the type of disease and mutation identified.

In a preferred embodiment, said mutation disease is a genetic disease such as cancer.

In a more preferred embodiment, the genetic disease is a cancer, preferably selected from the group consisting of: adenoma or primary tumors, such as colorectal cancer (also called colon cancer or large bowel cancer), colon adenocarcinoma, rectal adenocarcinoma, gastric cancer, stomach cancer, endometrial cancer, uterine cancer, uterine corpus endometrial carcinoma, breast cancer, bladder cancer, hepatobiliary tract cancer, liver hepatocellular carcinoma, urinary tract cancer, urothelial carcinoma, ovary cancer, ovarian serous cystadenocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma, bladder cancer, prostate cancer, kidney cancer, kidney renal papillary cell carcinoma, head and neck cancer, skin cancer, skin cutaneous melanoma, thyroid carcinoma, squamous cell carcinoma, lymphomas, leukemia, brain cancer, brain lower grade glioma, glioblastoma, glioblastoma multiforme, astrocytoma, and neuroblastoma, preferably pancreatic cancer, more preferably pancreatic ductal adenocarcinoma.

In a particular embodiment, said mutation detected in a patient nucleic acid (e.g. DNA) sample according to the method is a mutation within KRAS gene, preferably a mutation within the KRAS gene resulting in the mutation of a glycine at the position 12 or 13 of the KRAS protein, more preferably resulting in a KRAS G12D, G12V, G12C, G12A, G12S, G12R or G13D mutation, again more preferably KRAS G12D, G12V or G12C mutation and a decrease of the ribonuclease activity or the mutation allele frequency in the patient sample during the treatment is indicative that the patient is responsive to the therapeutic agent.

For example, when the mutation disease is a cancer, the therapeutic agent can be chemotherapy, targeted therapy or an immunotherapy agent.

Chemotherapy is a drug treatment that uses powerful chemicals to kill fast-growing cells in your body. Chemotherapy includes the use of cytotoxic anti -neoplastic agents, such as alkylating agents, anti-metabolites, anti-microtubule agents, Topoisomerase inhibitors, cytotoxic antibiotics and others. Examples of chemotherapeutic drugs include with no limitations: Capecitabine, 5-FU, docetaxel, SN-38, CPT11, cisplatin, carboplatin, etc.

Targeted therapy includes the use of “targeted” drugs such as small molecule inhibitors or neutralizing monoclonal antibodies, that target proteins that are abnormally expressed in cancer cells and that are essential for their growth such as for example receptor and non-receptor tyrosine kinases, growth factors, hormone receptors, and others.

Immunotherapy is a type of cancer treatment that activates the immune system to fight disease such as cancer. Immunotherapy that can be used to treat cancer includes as non-limiting examples: immune inhibitory checkpoint inhibitors which are drugs that block inhibitory immune checkpoint protein, T-cell transfer therapy, monoclonal antibodies or immune system activators. As used herein the term "immune checkpoint protein" has its general meaning in the art and refers to a molecule that is expressed by T cells and NK cells and regulates the immune system. According to the present disclosure, immune checkpoint proteins are preferably inhibitory immune checkpoint proteins that dampen effector immune response. Inhibitory immune checkpoint molecules are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g., Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al., 2011. Nature 480:480- 489).

The present disclosure relates to the use of a therapeutic agent in the manufacture of a medicament for the treatment of a mutation disease in a patient who is previously identified as having said disease in a method as described above.

In the context of the present disclosure, the term "treating" or "treatment", as used herein, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of the disorder or condition to which such term applies.

As used herein, a "therapeutically effective amount", “therapeutically efficient amount” or an "effective amount" means the amount of a composition that, when administered to a subject for treating a state, disorder or condition is sufficient to effect a treatment. The therapeutically effective amount will vary depending on the compound, formulation or composition, the disease and its severity and the age, weight, physical condition and responsiveness of the subject to be treated.

The therapeutic agent (e.g. cancer drug) described herein may be administered by any means known to those skilled in the art, including, without limitation, intravenously, orally, intra- tumoral, intra-lesional, intradermal, topical, intraperitoneal, intramuscular, parenteral, subcutaneous and topical administration. Thus, the compositions may be formulated as an injectable, topical, or ingestible formulation. Administration of the compounds or therapeutic agents to a subject in accordance with the present disclosure may exhibit beneficial effects in a dose-dependent manner. Thus, within broad limits, administration of larger quantities of the compositions is expected to achieve increased beneficial biological effects than administration of a smaller amount. Moreover, efficacy is also contemplated at dosages below the level at which toxicity is seen. It will be appreciated that the specific dosage of a therapeutic agent (e.g. cancer drug) administered in any given case will be adjusted in accordance with the composition being administered, the volume of the composition that can be effectively delivered to the site of administration, the disease to be treated or inhibited, the condition of the subject, and other relevant medical factors that may modify the activity of the compositions or the response of the subject, as is well known by those skilled in the art.

For example, the specific dose of a therapeutic agent (e.g. cancer drug) for a particular subject depends on age, body weight, general state of health, diet, the timing and mode of administration, the rate of excretion, medicaments used in combination and the severity of the particular disorder to which the therapy is applied. Dosages for a given patient can be determined using conventional considerations, e.g., by customary comparison of the differential activities of the compositions described herein and of a known agent, such as by means of an appropriate conventional pharmacological protocol. The compositions can be given in a single dose schedule, or in a multiple dose schedule.

Suitable dosage ranges for a therapeutic agent may be of the order of several hundred micrograms of the agent with a range from about 0.001 to 10 mg/kg, preferably with the range from about 0.01 to 1 mg/kg, more preferably from about 1 to 10 mg/kg, again more preferably 10 mg/kg.

A method for evaluating a treatment response in a patient having a mutation disease during the course of the treatment.

The detection of a mutation in a patient nucleic acid sample according to the present disclosure makes it possible to classify the patient as a responder of a therapeutic agent by determining the ribonuclease activity or the mutation allele frequency in a nucleic acid (e.g. DNA) sample and by determining whether the ribonuclease activity or the mutation allele frequency is increased or decreased during the treatment, preferably in comparison to the ribonuclease activity or the mutation allele frequency in a nucleic acid (e.g. DNA) sample obtained from said patient at a prior time point of the treatment, preferably prior to the administration of at least one therapeutic agent. The present disclosure relates to an in vitro method for evaluating the therapeutic response to a therapeutic agent in a patient having a mutation disease, said method comprising detecting a mutation in a nucleic acid (e.g. DNA) subject sample of a patient having received at least one dose of a therapeutic agent according to the method as described above, in particular by: a) selectively amplifying a target nucleic acid sequence comprising said mutation from said sample by allele-specific polymerase amplification (e.g. PCR or RPA), by contacting said nucleic acid sample to a pair of variant allele-specific primers as described above and a DNA polymerase, b) in vitro transcribing amplified nucleic acid sequence comprising said mutation into target RNA, c) contacting said target RNA with a Cas protein having ribonuclease activity (e g. Casl3a or a functional variant thereof), a guide RNA comprising a complementary sequence to a part of the target RNA comprising said mutation, and preferably an RNA reporter, and d) determining the ribonuclease activity, and optionally determining the mutation allele frequency in said sample, wherein a decrease of the ribonuclease activity or the mutation allele frequency in the patient sample during the treatment is indicative that the patient is responsive to the therapeutic agent.

The term "responder, or responsive to a therapeutic agent" refers to a subject in whom the onset of at least one of the symptoms of the condition to be treated is delayed or prevented, upon or after treatment, or whose symptoms or at least one of the symptoms stabilize, diminish or disappear.

“Therapeutic response” refers to the consequence of a medical treatment in a patient, the results of which are judged to be useful or favorable. For instance, a therapeutic response may be the delay or the prevention of at least one of the symptoms, upon or after treatment, or may be that symptoms or at least one of the symptoms in patient stabilize, diminish or disappear.

The term “evaluating therapeutic response to treatment”, as used herein, refers to an ability to assess whether the treatment of a patient is likely effective in (e.g., providing a measurable benefit or positive medical response to) the patient after some time of administration of the treatment.

As used herein, the term "control value" or “baseline value” may refer to ribonuclease activity or the mutation allelic frequency in biological sample obtained from said patient at a different time, preferably prior to said administration of therapeutic agent dose or prior to any administration of therapeutic agent dose or prior to any treatment of the condition or disease. The control value may alternatively be a predetermined value such as a threshold value, a standard value or a range obtained from other source than the patient’s data. The control predetermined value may be established based upon comparative measurements between patients prior to said administration of therapeutic agent dose and patients having received said therapeutic agent dose administration. In a preferred embodiment, the threshold value refers the ribonuclease activity or the mutation allele frequency in a biological sample obtained from said patient monitored at a different time, preferably at a prior time point of the treatment.

The therapeutic response can be evaluated according to the present method, before treatment and/or throughout the course of treatment for monitoring the therapeutic response over time.

According to the present method for evaluating therapeutic response, the sample is previously collected from a patient having received a dose of a therapeutic agent. According to the present disclosure, a patient having received a therapeutic agent refers to a patient having received at least one dose of a therapeutic agent. The therapeutic response can be evaluated according to the present method, after each administration of dose of a therapeutic agent throughout the course of treatment for monitoring the therapeutic response over time.

In a preferred embodiment, the ribonuclease activity or the mutation allele frequency is determined in a nucleic acid sample obtained from said patient at a different time, preferably prior to said administration of therapeutic agent dose or prior to any administration of therapeutic agent dose or prior to any treatment of the condition or disease or the control value refers to a control predetermined value established based upon comparative measurements between patients prior to said administration of therapeutic agent dose and patients having received said therapeutic agent dose administration. In a preferred embodiment, the ribonuclease activity or the mutation allele frequency is determined in a sample collected from a patient at least 1, 2, 3, or 7, preferably 10, 14, 21, 28 days after therapeutic agent dose administration, more particularly at least 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120 days after therapeutic agent dose administration.

The ribonuclease activity or the mutation allele frequency in a sample of patient having received at least one first dose of therapeutic agent during the treatment, preferably in comparison to the ribonuclease activity or the mutation allele frequency in a sample obtained from said patient at a prior time point of the treatment, preferably prior to the administration of at least one therapeutic agent, correlates with therapeutic response of said patient to therapeutic agent.

The method for evaluating therapeutic response according to the present disclosure can indicate success or failure of treatment to a patient.

A lower ribonuclease activity or a lower mutation allele frequency in a patient sample compared to a control value is indicative that the patient is responsive to said treatment. Typically, the ribonuclease activity or the mutation allele frequency is deemed to be lower than the control value if change in said patient to that of said control value is lower than at least 0.1, preferably 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, more preferably 1, 2, 3, 4 again more preferably 5.

A higher or similar ribonuclease activity or mutation allele frequency in a patient sample compared to a control value is indicative that the patient is non-responsive to said treatment. If after treatment with the therapeutic agent, the ribonuclease activity or the mutation allele frequency in sample of a patient having received at least one dose of the therapeutic agent is not lower than a control value, the treatment should be interrupted or modified.

In a particular embodiment, said mutation disease is a cancer and the treatment may be a cancer drug such as chemotherapy, targeted therapy or an immunotherapy agent as described above.

In a particular embodiment, said mutation detected in a patient nucleic acid (e.g. DNA) sample according to the method is a mutation within KRAS gene, preferably a mutation within the KRAS gene resulting in the mutation of a glycine at the position 12 or 13 of the KRAS protein, more preferably resulting in a KRAS G12D, G12V, G12C, G12A, G12S, G12R or G13D mutation, again more preferably KRAS G12D, G12V or G12C mutation and a decrease of the ribonuclease activity or the mutation allele frequency in the patient sample during the treatment is indicative that the patient is responsive to the therapeutic agent, in particular cancer drug such as chemotherapy, targeted therapy or an immunotherapy agent as described above.

Kit

The present disclosure also encompasses a kit for identifying a sequence variant (e.g., mutation, preferably KRAS gene mutation) in a nucleic acid (e g. DNA) sample.

According to the present disclosure, said kit comprises: a) a first variant allele specific primer, preferably comprising a sequence complementary to a part of the target nucleic acid sequence such that the 3 ’-end of the primer binds to the sequence variant (e g., mutation) in the nucleic acid target sequence, and optionally further comprises a mismatch with the target nucleic acid sequence, more preferably at the antepenultimate base of the first primer, b) a second variant allele specific primer comprising a sequence complementary to a part of the target nucleic acid and preferably further comprises a bacterial RNA polymerase promoter such as T7 promoter, T3 promoter or SP6 promoter, c) a guide RNA comprising a complementary sequence to a part of the target RNA comprising said sequence variant (e.g., mutation), and, optionally d) a Cas protein having ribonuclease activity, preferably Cast 3a protein or a functional variant thereof.

In a preferred embodiment, said kit comprises: a) a first variant allele specific primer, preferably comprising a sequence complementary to a part of the target nucleic acid sequence such that the 3 ’-end of the primer binds to the sequence variant (e g., mutation) in the nucleic acid target sequence, and optionally further comprises a mismatch with the target nucleic acid sequence, more preferably at the antepenultimate base of the first primer, b) a second variant allele specific primer comprising a sequence complementary to a part of the target nucleic acid and preferably further comprises a bacterial RNA polymerase promoter such as T7 promoter, T3 promoter or SP6 promoter, c) a guide RNA comprising a complementary sequence to a part of the target RNA comprising said sequence variant (e.g., mutation), and, d) a Cas protein having ribonuclease activity, preferably Cast 3a protein or a functional variant thereof.

The kit may further comprise an RNA reporter, preferably comprising a fluorophore and a quencher and/or bacterial RNA polymerase, preferably selected from the group consisting of: T7 RNA polymerase, T3 RNA polymerase or SP6 polymerase.

In a preferred embodiment, the present disclosure relates to a kit for detecting a mutation within KRAS gene resulting in KRAS^G12D mutation in a nucleic acid (e.g. DNA) sample comprising: a) a first AS primer comprising or consisting of a nucleic acid sequence of SEQ ID NO: 2, b) a second AS primer comprising or consisting of a nucleic acid sequence of SEQ ID NO: 3, preferably SEQ ID NO: 5, c) a guide RNA comprising or consisting of a nucleic acid sequence of SEQ ID NO: 7 and optionally, d) a Cas protein having ribonuclease activity, preferably Casl3a protein or a functional variant thereof. The kit may further comprise an RNA reporter, preferably comprising a fluorophore and a quencher and/or bacterial RNA polymerase, preferably selected from the group consisting of: T7 RNA polymerase, T3 RNA polymerase or SP6 polymerase.

In some embodiments, said kit can further comprise a DNA polymerase and/or dNTP. Thermostable DNA polymerases are typically described in Newton and Graham 1994 In: PCR, BIOS Scientific Publishers, Ltd., Oxford, UK. 13.

Primers, guide RNA, target sequence usable in a kit according to the disclosure have been previously described.

The kit as above mentioned can be used in the clinical applications as previously described.

The invention will now be exemplified with the following examples, which are not limitative, with reference to the attached drawings in which:

Examples

1. Materials and Methods

Reagents and enzymes

LwaCasl3a enzyme was obtained from GenScript and stored at -80°C in 50 mM Tris-HCl, 600 mM NaCl, 5% Glycerol, 2 mM DTT, pH 7.5. PAGE Ultramer DNA oligos for RNA guide synthesis were supplied by Integrated DNA Technologies (IDT, United States). HiScribe™ T7 Quick High Yield RNASynthesis Kit, containing T7 polymerase, RNAse inhibitor, and NTP mix buffer was obtained from New England Biolabs (NEB, United States). PCR primers were obtained from Eurogentec (Belgium). Hydroxyethyl piperazine ethane sulfonic acid (HEPES) and dimethylsulfoxide (DMSO) were supplied by Sigma-Aldrich (United States).

Cell culture

BxPC-3, AsPC-1, and MIA PaCa-2 cells were maintained in Dulbecco’s Minimal Essential Medium (DMEM, Invitrogen, Saint Aubin, France), Capan-1 cells were maintained in Roswell Park Memorial Institute (RPMI, Invitrogen). For both media, 10% Fetal Bovine Serum (FBS, Invitrogen), 100 U/mL penicillin (Invitrogen), and 100 pg/mL streptomycin (Invitrogen) were added. All cell lines were cultured at 37 °C, 5% CO2 in a humidified chamber.

Patient S inclusion and sample collection

Patients were recruited prospectively between February 2023 and March 2023. All patients who received endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) in the context of pancreatic mass during these 2 months were recruited. Patients filled out an informed consent before EUS-FNA. This work was performed following the human and ethical principles of research outlined in the Helsinki guidelines and following local statutory requirements (Acceptance of the study by the Bordeaux University Hospital ethics review board on 27/09/2023, reference CER-BDX 2023-102).

DNA extraction and quantification

DNA samples for KRAS detection were extracted from pancreatic tumor cell lines using QIAamp DNA extraction kit® (Qiagen, France). KRAS^WT/WT DNA, KRA^^I2D/GI2D, KRAS^G2, '^<G2 - and X/M,S^{<il 2V GI 2V} mutant DNA were extracted from BxPC-3, AsPC-1, MIA PaCa-2, and Capan-1 cell lines respectively, and verified by NGS analysis using the Bordeaux University Hospital Tumor Biology Department routine solid tumor panel (custom ampliseq panel with Ion Torrent S5 sequencer (Thermo Fisher Scientific, United States)). All DNA samples were quantified by spectrophotometry using Nanodrop® One/One device (Thermo Fisher Scientific, United States). For molecular analysis on needle-rinsing fluids, Maxwell RSC ccfDNA Plasma Kit was used for DNA extraction and DS11FX automated system (DeNovix) for concentration evaluation.

RNA guide synthesis and purification

Guide RNAs were produced by T7-mediated in vitro transcription as described in Kellner et al. (Kellner MJ, et al. Nat. Protoc. 2019 ; 14 : 2986-3012). Briefly, oligonucleotides (PAGE Ultramer DNA oligos from Integrated DNA Technologies) were resuspended at a concentration of 100 pM. Annealing was performed at 95°C for 5 minutes followed by a slow temperature decrease to 4°C (0.1°C/s), using common forward p.T7 oligo and Taq buffer 10X. In vitro transcription was next performed overnight with the Hi Scribe™ T7 Quick High Yield RNA Synthesis Kit (NEB, MA, USA) following the manufacturer’s instructions and subsequently purified with Agencourt RNAClean XP beads (Beckman Coulter). Purified RNA products were aliquoted and frozen at -80°C.

DNA amplification step

PCR and allele-specific PCR amplifications were performed using Phire Tissue Direct PCR Master Mix® (Thermo Fisher Scientific) following the manufacturer's instructions. Amplification primers and related annealing temperatures are listed in Table 2.

Table!: Primers and crRNAfor KRAS⁰¹² specific and non-specific amplification and detection. The sequence of T7 promoter (underlined) framed by 5 extra-bases (grey) and three G nucleotides (bold) for improved in vitro transcription are shown.

All amplifications were performed using 10 ng of gDNA input, except for patient samples with non-sufficient DNA concentration. An overhang including the T7 promoter was used to enable subsequent T7-mediated in vitro transcription of the PCR products (Kellner MJ, et al. Nat. Protoc. 2019 ; 14 : 2986-3012). All primers were used at a concentration of 250nM. By default, 35 cycles of amplification were performed. For CASPER, and after multiple conditions testing, only 30 cycles of amplification were performed to optimize specificity. CRISPR-Casl3a detection step

RNA guide spacer sequences are listed in Table 2. In vitro transcription of KRAS PCR products and Casl3-mediated detection of T7-produced RNA were performed simultaneously as described previously (Kellner MJ, et al. Nat. Protoc. 2019 ; 14 : 2986-3012). The detection mix included 16 mM HEPES, 7.2 mM MgCl₂, 640 nM rNTP, 0.05 U.pL’¹ T7 RNA polymerase, 1.6xl0³ U.pL'¹ murine RNase inhibitor (NEB), 5 pg.pL'¹ LwaCasl3a protein, 400 pg.pL'¹ RNA guide, 100 nM fluorescent RNA reporter. The final volume of the reaction was 20 pL including 1 pL of PCR products. All manipulations were performed on ice. After the addition of PCR products, samples were immediately transferred to a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) and the fluorescence level was quantified every minute for 90 minutes. Results analysis was performed using CFX MaestroTM software (BioRad). The fluorescence intensity ratio was calculated at 90 minutes as follows:

(Mutant template fluorescence at 90 min — Mutant template fluorescence at 1 min) (WT template fluorescence at 90 min — WT template fluorescence at 1 min)

Real-time quantitative PCR

Detection of 7dN'^{l 2IJ} and KRAS^m alleles was performed using the Promega GoTaq® qPCR kit (Promega, Wisconsin) following the manufacturer's instructions using lOng DNA input. The primers used and annealing temperatures are summarized in Table 2. All primers were used at a concentration of 2.5pM. By default, 35 cycles of amplification were performed. Data were analyzed with CFX Maestro Software (Bio-Rad). Relative expression ot' KRAf''^2,J and KRAS^WT alleles was first normalized to GAPDH expression and then represented as fold changes (2'^AACt). Melting curves showed that primers amplified only the specific fragments.

Droplet digital PCR

Droplet digital PCR analyses were performed on the BioRad ddPCR platform (BioRad, United States) with a QX-200 TM droplet generator and a QX-200 TM droplet reader. Biorad KRAS G12/G13 Screening and Biorad KRAS G12D-specific kits were used for the global or specific KRAR ’¹² mutant detection, according to the manufacturer's instructions. To compare the performance of ddPCR vs. PCR-CRISPR-Casl3a or CASPER, all experiments were performed using 10 ng of DNA. For patients’ samples, various amount of DNA (18 pL, regardless of DNA concentration) were used for KRAS G12/13 multiplex ddPCR screening assay, and 10 ng were used for KRAS G12D specific ddPCR assay, except for patient samples with non-sufficient DNA concentration. Results analysis was performed using QuantasoftTM software (BioRad) with lab-validated clinical routine interpretation guidelines. For each assay, & wild-type sample and a “no DNA input” control were analyzed. MAE positivity threshold (0.056%) was previously determined (Buscail E, et al. Cancers 2019 ; 11 : 1656). A sample was considered positive when the lower standard deviation value of the MAF was greater than the positivity threshold.

RNA secondary structures analysis

Predicted secondary RNA structures of KRAS T7 RNA products were obtained with RNAfold® software (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). Default parameters were used.

Statistical analysis

Statistical tests were performed using the Graph-Pad Prism software (v6.04). Results are expressed as mean ± SEM or mean ± SD, analyzed by unpaired, bilateral Student's t-tests with Welch’s correction. Correlation analyses were performed using Spearman’s test. p<0.05 was considered statistically significant.

2. Results

2.1 KRAS allele discrimination by CRISPR-Casl3a using crRNA guides hybridizing the mutant nucleotide on position 19 of the spacer sequence

The inventors first used the CRISPR-Casl3a platform to detect the most frequent alleles, KRAS^^12D, KRA^^12V’ and KRAS°^12C using a crRNA design as previously documented to efficiently target KRAS' ^m mRNAs in cellulo (Zhao X, Liu L, Lang J, et al. Cancer Lett. 2018 ; 431 : 171-181). The inventors first tested the in vitro discrimination ability of crRNA19^G12X guides (with discriminative nucleotide position placed on the 19^th nucleotide of the spacer crRNA sequence), perfectly matching the mutant allele and presenting one mismatch with the WT allele. Casl3a collateral RNase activity on reporter fluorescent RNA probes was induced by all 3 guides but the guides also hybridized the WT allele (Figure la-c, bars and curves). Noticeably, discrimination variations were observed between the guides, the crRNA19^G12C bearing the best specificity, with a maximal fluorescence intensity ratio to WT detection of 11.5±3.5 times (versus 2.1±0.5 for crRNA19^G12V and 1.5±0.1 for crRNA19^G12D).

Due to the low specificity observed in the initial basic crRNA design, the inventors introduced a mismatch at position 14, to obtain the crRNA19^G12X-14, presenting 1 mismatch with the mutant allele and 2 mismatches with the WT allele. The synthetic mismatch at position 14 slightly improved the detection of KRAS³¹²³² (maximal fluorescence intensity ratio of 2.2±0.3 versus 1.5±0.1), did not change the detection of KRA^ ^c (maximal ratio of 9.9±1.8), but diminished the detection of KRAS³²' (1.4±0.1 versus 2.1±0.5) (Figure 2a-c).

As the addition of one synthetic mismatch impacted differently the ITT/mutation discrimination, the inventors produced a guide with 2 synthetic mismatches with the mutant 7 S^{<,I 2D} allele and 3 mismatches with the WT allele. Specificity was unchanged for the crRNA19^G12D-14-18 (fluorescence ratio of 2.3±0.1). Thus, although position 19 may distinguish KRAS'³³ from KRAS^WT with some specificity, the discrimination of WT and KRAS³¹²¹³ alleles is not sufficient, even when 3 mismatches were present between the crRNA guide and the WT template.

2.2 KRAS allele discrimination by CRISPR-Casl3a using crRNA guides hybridizing the mutant nucleotide on position 12 of the spacer sequence

The “seed” region of the crRNA spacer sequence, covering nucleotides 9 to 15, is described as more sensitive to mismatches (Gootenberg JS, et al. Science 2017; 356 : 438-442). The inventors thus designed the crRNA12^G12D. Specificity was slightly improved compared to the crRNA19^G12D (maximal fluorescence intensity ratio to WT signal of 2.0±0.2 versus 1.5±0.1), but was not sufficient for full discrimination (Figure 3a). Indeed, using CRISPR-Casl3a for low-frequency mutant allele detection implies the absence of reporter RNA cleavage by the Casl3a with the WT template. Introduction of synthetic mismatches (crRNA12^G12D-13 and crRNA12^G12D-13-l 1) did not fully discriminate alleles (maximal fluorescence ratio to WT signal of 1.6±0.05 and 2.0±0.6, respectively) and resulted in a global loss of signal for the crRNA12^G12D-13-ll (Figure 3b, c). The inventors also tested if a mismatch at the 5' extremity of the spacer sequence could diminish the recognition of the KRAS³³ allele and designed the crRNA4^G12D. As for the crRNA 19^G12D, the crRNA4^G12D did not present any specificity for the KRAS³¹²⁰ allele (maximal fluorescence intensity ratio of l.l±0.1).

The inventors next challenged the crRNA 12^G12D guide with sensitivity experiments on serial DNA dilution samples. CRISPR-Casl3a sensitivity was 10%, while a conventional ddPCRwas 10-fold more sensitive (Figure 3d). 2.3 KRAS allele discrimination by CRISPR-Casl3a using hairpin spacer crRNA guides

Hairpin-spacer crRNA guides feature an additional sequence downstream of the spacer, which competes for hybridization with the spacer either on the target DNA (mutant or WT) or with the spacer itself This competition, aided by hairpin structures, may minimize binding to the WT allele while maintaining sufficient binding to the mutant allele (Ke Y, Huang S, Ghalandari B, et al. Adv. Sci. 2021 ; 8 : 2003611). The inventors designed, with discriminative nucleotide position still placed on the 12^th nucleotide, 3 different hairpin-spacer crRNAs with or without additional synthetic mismatches. The hairpin-spacer guides were not able to fully discriminate KRAS alleles (Figure 4a-c) or to increase sensitivity over that of the crRNA12^G12D (Figure 4d).

2.4 CASPER: Coupling CRISPR-Casl3a sensitivity and Allele-specific PCR specificity

Among the CE-//1 vitro diagnostic (CE-IVD) platforms offering highly specific identification of single nucleotide variants (SNPs) and besides ddPCR, allele-specific (AS) based methods provide good performance (Lazaro A et al. ACS Sens. 2022 ; 7 : 758-765). However, the limit of detection highly depends on the DNA input (Milbury CA, et al. Biomol. Detect. Quantif. 2014 ; 1 : 8-22). Here, the inventors tested the potential of CRISPR-Casl3a for the identification of low-frequency KRAS mutant alleles in a limited DNA quantity (10 ng), compatible with liquid biopsy circulating-free DNA (cfDNA) analysis or other applications with low DNA input. The routinely-used AS-based method is qPCR. The determination of sample positivity in qPCR depends on the cycle at which amplified DNA is first detected, following method validation and interpretation guidelines. This is particularly crucial in addressing non-specific amplifications that may occur at high cycle numbers (Nolan T, et al. Good practice guide for the application of quantitative PCR (qPCR). 2013). The inventors hypothesized that in this challenging low range of mutant allele frequency, the sensitivity of CRISPR-Casl3a for the detection step may make the difference. Thus, AS regular PCRs were carried out, with the 3’ nucleotide-specific primer hybridizing the mutant nucleotide, and carrying an additional synthetic mismatch to inhibit the amplification of the WT allele (Figure 5a). AS-PCR was followed by CRISPR-Casl3a detection with a crRNA-AS^G12D covering 15 nucleotides of the AS primer and 13 nucleotides of the amplified sequence (CASPER, Figure 5b). The fluorescence profile revealed a full discrimination between mutant and WT alleles (Figure 6a). The maximal fluorescence ratio to the WT signal was 22.9±8.8. This high discrimination translated into a high sensitivity of 0.5% (Figure 6b), superior to conventional allele-specific PCR (Figure 5c) and equal to ddPCR (Figure 5d). Moreover, the present strategy was quantitative for a MAF of up to 10%. A 4-parameter fit curve could be built and a Spearman correlation analysis showed a significant p value (^=0.78 and r=0.785 with p=0.02, respectively; Figure 5 e, f).

2.5 CASPER for the detection of the KRAS^G12D mutation in pancreatic cancer patients’ liquid samples

To demonstrate the clinical feasibility of this assay, the inventors analyzed fine needle aspiration fluids from 24 patients suspected of PDAC (Figure 6c). All samples were first tested for KRAS G12/G13 mutations by ddPCR in laboratory routine conditions with the multiplex KRA ^{GV2JG 3} mutation kit, using conventionnal DNA input. Eighteen samples (18/24, 75%) were found positive for one of the 7 KRAS^{G ,Gi} mutations covered by the assay. Using CASPER assay on the same 24 samples, the inventors detected 6 KRASP^12D positive samples (Figure 6 d, e). The DNA inputs varied from 6 to 10 ng depending on the initial sample DNA concentration. All of the 6 samples were also positive for ddPCR multiplex KRASP '^G13 mutation test, with mutant allele frequencies ranging from 0,43% to 48.7%. To confirm the CASPER specific /\7MS^<,I2I) mutation detection, the inventors tested 24 samples using a specific KRAS G12D ddPCR assay and identified 5 positive samples. Noticeably, sample 6, that presented the lower allelic frequency in the first ddPCR assay (0,43%, 39.7 ng DNA input), was positive with CASPER and negative with specific ddPCR, using the same input of 10 ng DNA (red arrow). Finally, a 4-parameter fit curve analysis of the fluorescence intensities ratio and ddPCR-based KRAS°^l2D mutation allelic frequency showed that the CASPER assay was fully quantitative (r²=0.98).

3. Discussion

PDAC management is hindered by a lack of effective treatments and challenges in swiftly confirming tumor presence. This study the first to test the ability of a conventional CRISPR- Casl3a platform's ability to discriminate KRAS'’ ¹ and KRAS^MUT alleles to detect low-frequency point mutations using a limited DNA input. For this purpose, methods with high specificity and sensitivity are needed. The inventors first used crRNA guides with the 19^th nucleotide of the spacer hybridizing the mutant position (Zhao X, et al. Cancer Lett. 2018 ; 431 : 171-181). For in vitro KRAS allele discrimination purposes, the position of the mismatch with the WT allele, out of the seed region, was not ideal. Non-specificity was also observed in cellulo (Zhao X, et al. Cancer Lett. 2018 ; 431 : 171-181), and the addition of another mismatch at position 14 marginally improved specificity. Guides mismatching the WT allele at positions 12 and 4 of the spacer did not allow full discrimination either. By testing different G12 mutation positions, these findings confirmed that a single mismatch in the guide's spacer sequence distinctly influenced specificity (Abudayyeh OO, et al. Science 2016 ; 353 : aaf5573; Kellner MJ, et al. Nat. Protoc. 2019 ; 14 : 2986-3012). Indeed, heterocyclic purine/purine mismatches may create a local steric bulk affecting more the crRNA hybridization to the K AS''' ¹ template than purine/pyrimidine mismatches (Rossetti G, et al. Nucleic Acids Res. 2015 ; 43 : 4309-4321). However, crRNA^G12D (G/U mismatch on the WT allele) and crRNA^G12V (G/A mismatch) showed similar profiles, while crRNA^G12C (G/A) was more discriminant, confirming that the purine or pyrimidine status of the mismatch-related bases cannot explain alone the specificity variations. A recent study revealed another level of complexity showing that mismatch type, for example, A-G displayed various specificities according to the position of the guanine nucleotide on the guide or the template (New design strategies for ultra-specific CRISPR-Casl3a-based RNA detection with single-nucleotide mismatch sensitivity, Nucleic Acids Research, Oxford Academic n.d). The neighboring sequence likely contributes to specificity modulation (New design strategies for ultra-specific CRISPR-Casl3a-based RNA detection with single-nucleotide mismatch sensitivity, Nucleic Acids Research, Oxford Academic n.d). Moreover, concentrations of templates and RNA reporters could influence the fluorescence kinetics of Casl2 and Casl3 detection systems (Huyke DA, et al. Anal. Chem. 2022). During the specificity assays, the inventors used saturating amounts of DNA templates (WT or mutant) and RNA reporters. Thus, they believe that the Vmax only depends on the ability of the guide to efficiently hybridize the template.

KRAS RNA templates adopt secondary structures leading to the formation of slightly different hairpin loops, rendering the sequence complementary to crRNAs more or less accessible. The G12C mutation, which was best discriminated from the WT sequence is in a stem, whereas the other mutations or the WT nucleotides are in loops. In addition, target RNA secondary structures may also need more energy for crRNA hybridization augmenting the global Gibbs free energy of Casl3a activation (Ke Y, Huang S, Ghalandari B, et al. Adv. Sci. 2021 ; 8 : 2003611). This suggests that each template/guide couple may display distinct energetic properties limiting the generalization of guidelines for crRNA design. Indeed, crRNA19^G12D and crRNA4^G12D were less discriminant than crRNA12^G12D. When comparing the positioning of the 3 guides on the KRAS° ^D RNA, the inventors observed that secondary structures may affect crRNA hybridization. The crRNA4^G12D and the crRNA19^G12D hybridization zones fully covered stemloop structures (lateral or terminal), which are only partly involved in the hybridization of crRNA12^G12D. The additional energy required to separate the loop structure could therefore also affect crRNA hybridization, impacting target RNA detection (Ke Y, Huang S, Ghalandari B, et al. Adv. Sci. 2021 ; 8 : 2003611). Recent studies report that spacer sequence length also greatly influences crRNA hybridization and recognition properties (New design strategies for ultraspecific CRISPR-Casl3a-based RNA detection with single-nucleotide mismatch sensitivity, Nucleic Acids Research, Oxford Academic n.d.). While the present work respected conventional 28-nucleotide spacer sequences, decreasing the spacer length could potentially serve as a solution to limit non-specific crRNA recognition. Of note, the link between crRNA guide hybridization and Casl3a activation is also complex: a strong hybridization of the crRNA with its target will not necessarily lead to strong activation of the nuclease, and vice versa (Tambe A, et al. Cell Rep. 2018 ; 24 : 1025-1036). Finally, 2 or 3 mismatches between the crRNA guide spacer and the target WT KRAS RNA sequence did not prevent non-specific hybridization and Cas activation.

Specificity was achieved by combining AS-PCR preamplification with a CRISPR-Casl3a detection. Fluorescence signal amplification, possible with Casl3a activation, made it possible to identify lower levels of PCR amplification, compared with conventional dye-based qPCR. CASPER achieved similar or better sensitivity than ddPCR with low DNA input (6-10 ng), detecting one KRAS^<,nv> ddPCR-false-negative patient. CASPER requires a conventional thermocycler with fluorescence detection, making it easy to implement as complementary to standard assays, especially when dealing with limited DNA input. KRAS'¹² detection with high sensitivity but imperfect specificity was achieved by the Cas 12 enzyme after a PCR preamplification (Zhou H, et al. Diagn. Basel Switz. 2021 ; 11 : 125). The initial amount of DNA input was not shared. In conclusion, facing the low specificity and sensitivity of the CRISPR-Casl3a for the detection of KRAS G12 point mutations, the inventors implemented the CASPER assay that enables specific and sensitive detection of KRAS^mut alleles. They proved that this strategy is equal or superior to ddPCR when using a small amount of input DNA, thus compatible with challenging low DNA quantity samples. CASPER versatility residing in the easy crRNA guide design and costless equipment could be a valuable solution to produce personalized molecular tools, implementable to “hotspots” or less common mutations.

Claims

1. A method of detecting a sequence variant within a nucleic acid sample, preferably DNA sample, wherein said method comprises: a) selectively amplifying a target nucleic acid sequence comprising said sequence variant from said sample by allele-specific polymerase amplification, preferably polymerase chain reaction amplification (PCR) or recombinase polymerase amplification (RPA), by contacting the sample to a pair of variant allele-specific primers and a DNA polymerase, a) in vitro transcribing amplified target nucleic acid sequence comprising said sequence variant into target RNA, b) contacting said target RNA with a Cas protein having ribonuclease activity and a guide RNA comprising a complementary sequence to a part of the target RNA comprising said sequence variant, c) determining the ribonuclease activity, and d) optionally determining the allelic frequency of said sequence variant in said sample.

2. The method of claim 1 wherein a first AS primer comprises a sequence complementary to a part of the target nucleic acid sequence such that the 3 ’-end of the first primer binds to the sequence variant in the nucleic acid target sequence.

3. The method of claim 2 wherein said first AS primer further comprises a mismatch with the target nucleic acid sequence, preferably at the antepenultimate base of the first AS primer.

4. The method according to claim 2 or 3 wherein the second AS primer comprises a sequence complementary to a part of the target nucleic acid sequence and further comprises a bacterial RNA polymerase promoter such as T7 promoter, T3 promoter or SP6 promoter and wherein the in vitro transcription of step b) is performed by adding a bacterial RNA polymerase, preferably selected from the group consisting of: T7 RNA polymerase, T3 RNA polymerase or SP6 polymerase.

5. The method according to any one of claims 1 to 4 wherein said Cas protein having ribonuclease activity is Casl3a protein or functional variant thereof.

6. The method according to any one of claims 1 to 5 wherein an RNA reporter, preferably comprising a fluorophore and a quencher is added in step c) and fluorescence intensity is measured in step d) to determine the ribonuclease activity.

7. The method according to any one of claims 1 to 6 wherein said sample is tumor nucleic acid, preferably tumor DNA comprising less than 20 ng of nucleic acid, more preferably tumor DNA in biological fluids.

8. A method for identifying a subject having a mutation disease comprising detecting said mutation in a nucleic acid sample from said subject according to a method of any one of claims 1 to 7, and wherein a higher ribonuclease activity or a higher mutation allelic frequency as compared to a corresponding threshold value is indicative that the subject has or is susceptible to have said disease.

9. The method according to claim 8 wherein said mutation disease is cancer, preferably selected from the group consisting of: pancreatic, colorectal, lung, ovarian and urogenital cancer, more preferably pancreatic cancer.

10. The method according to claim 9 wherein said mutation is within an oncogene, preferably KRAS gene, more preferably wherein said mutation results in the mutation of a glycine at the position 12 or 13 of the KRAS protein, more preferably resulting in a KRAS G12D, G12V, G12C, G12A, G12S, G12R or G13D mutation.

11. A therapeutic agent for use in the treatment of a mutation disease in a subject in need thereof, wherein said therapeutic agent is administered in a subject previously identified as having a mutation disease using any method according to any one of claims 8 to 10.

12. A method for evaluating a therapeutic response in a patient having a mutation disease, comprising detecting a mutation in a nucleic acid sample according to the method of any one of claims 1 to 7, wherein a decrease of the ribonuclease activity or the mutation allelic frequency during the treatment is indicative that the patient is responsive to the therapeutic agent.

13. The method of claim 12 wherein said disease is a cancer, preferably selected from the group consisting of: pancreatic, colorectal, lung, ovarian and urogenital cancer, more preferably pancreatic cancer, and more preferably wherein said mutation is within an oncogene, preferably KRAS gene, more preferably wherein said mutation is KRAS G12 or G13 mutation, more preferably selected from the group consisting of: KRAS G12D, G12V, G12C, G12A, G12S, G12R and G13D mutation.

14. A kit for detecting a sequence variant in a nucleic acid sample comprising: a) a first allele specific primer, preferably comprising a sequence complementary to a part of the target nucleic acid sequence such that the 3 ’-end of the primer binds to the sequence variant in the nucleic acid target sequence, and optionally further comprises a mismatch with the target nucleic acid sequence, more preferably at the antepenultimate base of the first primer, b) a second allele specific primer comprising a sequence complementary to a part of the target nucleic acid and preferably further comprises a bacterial RNA polymerase promoter such as T7 promoter, T3 promoter or SP6 promoter, c) a guide RNA comprising a complementary sequence to a part of the target RNA comprising said sequence variant, and, optionally d) a Cas protein having ribonuclease activity, preferably Casl3a protein or a functional variant thereof.

15. The kit of claim 14 for detecting a mutation within KRAS gene resulting in KRAS^G12D mutation in a nucleic acid sample comprising: a) a first AS primer comprising or consisting of a nucleic acid sequence of SEQ ID NO: 2, b) a second AS primer comprising or consisting of a nucleic acid sequence of SEQ ID NO: 3, preferably SEQ ID NO: 5, c) a guide RNA comprising or consisting of a nucleic acid sequence of SEQ ID NO: 7 and optionally, d) a Cas protein having ribonuclease activity, preferably Cast 3a protein or a functional variant thereof.