CN116286734B

CN116286734B - Mutant of wild LbCAs12a protein and SNP detection application

Info

Publication number: CN116286734B
Application number: CN202211514174.0A
Authority: CN
Inventors: 张楹; 张红霞; 殷昊
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2022-11-29
Filing date: 2022-11-29
Publication date: 2024-04-02
Anticipated expiration: 2042-11-29
Also published as: CN116286734A

Abstract

The application discloses a mutant of a wild type LbCAs12a protein and SNP detection application thereof, wherein the amino acid sequence of the wild type LbCAs12a protein is shown as a seq_1, and the mutant has mutation at least at the 174 th position of the amino acid sequence shown as the seq_1. Compared with the wild LbCAs12a protein, the mutant has SNP identification capability, and based on the CRISPR-Cas system designed by the mutant, the SNP in the genome can be identified within 30 minutes, and the sample preparation process of the system is very simple, so that cross contamination can be avoided, and the operation difficulty and detection cost can be reduced.

Description

Mutant of wild LbCAs12a protein and SNP detection application

Technical Field

The application belongs to the field of biology, and particularly relates to a mutant of a wild LbCAs12a protein and SNP detection application thereof.

Background

Single Nucleotide Polymorphisms (SNPs) mainly comprise single base transitions, base transversions, base deletions or base insertions, are the most common type of heritable variation in the human genome, with an average of one SNP per 1-1.9 kb. SNPs are involved in many biological processes including genetic diseases, tumorigenesis, drug metabolism, etc. Pharmacogenomics SNPs can be divided into four categories of SNPs related to drug absorption, drug transport, drug metabolism and drug excretion according to their functions, and are closely related to the sensitivity of individuals to drug treatment. The cytochrome P450 (CYP) family is the most important drug metabolizing enzyme, mediating multiple drug metabolisms. There are more than two thousand CYP single nucleotide polymorphisms reported so far, some of which have a significant impact on their enzymatic activity. For example, CYP2C9 is one of the key enzymes involved in the metabolism of the anticoagulant drug warfarin, and the use of warfarin requires an accurate personalized prescription that achieves a precise balance between over anticoagulation (thrombosis) and under anticoagulation (hemorrhage). Two common variants of CYP2C9, namely CYP2C9 x 2 (3608C > t) and CYP2C9 x 3 (42614 a > C), were found to result in reduced enzyme activity. Heterozygous or homozygous individuals carrying the above mutations are more sensitive to warfarin than normal individuals, and therefore they are at greater risk of bleeding during warfarin treatment and therefore require lower doses. For another example, CYP2C19 is a key metabolizing enzyme of clopidogrel. Clopidogrel is an effective anti-platelet aggregation drug, which is metabolized by CYP2C19 to produce 2-oxo-clopidogrel and finally metabolized into active thiol metabolites to play a role in inhibiting platelet aggregation. Patients carrying CYP2C19 (6815 > A) alleles in their genome have lost CYP2C19 enzymatic activity in their bodies, and therefore, treatment with clopidogrel can result in ineffective treatment, delayed disease, and bleeding risk. Therefore, rapid and accurate detection of key pharmacogenomics SNPs is critical for personalized medicine when using high risk drugs. Since CYP2C9 and CYP2C19 play an important role in the metabolic processes of various medicines such as warfarin, clopidogrel, phenytoin sodium, sinbocmod, valproate and the like, it is very important to develop a rapid and accurate detection method by taking CYP2C9 and CYP2C19 as detection targets.

Currently, various SNP detection methods have been developed, and can be broadly classified into two major categories, that is, electrophoresis-based methods and high-throughput identification methods. SNP detection based on electrophoresis, such as denaturing gradient gel electrophoresis, allele-specific PCR, cleavage amplification polymorphism sequence marking and the like, has the characteristics of long time consumption, low accuracy, and capability of identifying but not identifying specific SNP types. High throughput identification methods, such as DNA sequencing and high resolution melting, have the advantage of high accuracy, but they are also time consuming and relatively expensive.

CRISPR-Cas systems are widely used in the fields of genome editing and nucleic acid detection, etc., where type V and VI Cas proteins, such as Cas12a, cas12b, cas13a and Cas14, etc., are capable of initiating non-specific trans-cleavage upon generation of specific cis-cleavage, a feature which is the basis of CRISPR detection systems. These Cas proteins are able to recognize and cleave target DNA sequences under the guidance of crrnas, which in turn produce nonspecific trans-cleavage activity, beginning to cleave single-stranded DNA or RNA. By utilizing the above characteristics, amplification of the detection signal can be achieved by adding a reporter DNA or RNA sequence to the detection system. In the Cas proteins described above, cas13a and Cas14 are able to distinguish SNPs in RNA or single stranded DNA substrates, respectively. However, since amplification of RNA substrates and single-stranded DNA substrates requires a reverse transcription or single-stranded DNA generation step, the method cannot be applied to a one-step detection system; mutations are introduced in the crRNA of Cas12b, which can distinguish SNPs in double stranded DNA substrates, but their optimal cleavage temperature is 48 ℃, which greatly limits their compatibility with isothermal amplification techniques. Because the optimal reaction temperature of the recombinase polymerase amplification technology (RPA) is 37-42 ℃, the Cas12b must be matched with the loop-mediated isothermal amplification (LAMP), the optimal reaction temperature of the LAMP is 55-65 ℃, the change of the reaction temperature can reduce the cleavage activity of the Cas12b and prolong the incubation time, and finally the detection speed is reduced.

In contrast, cas12a can be perfectly matched with RPA, and Cas12a uses double-stranded DNA as a substrate, widely applied to nucleic acid detection. However, the need for TTTV-PAM and low specificity limit its use in SNP detection. Even if crRNA is extended, including adding additional TA-rich DNA sequences at the 3 'end of the crRNA, inserting nucleotides at the spacer (nucleotides at the 3' end of the crRNA and spacer) does not distinguish SNPs in the substrate well. Cas12a can distinguish SNPs in a two-step reaction by adding classical PAM sequences upstream of the target site and introducing additional mismatches on the crRNA during PCR amplification. Recent studies have disclosed a one-step nucleic acid detection system based on LbCas12a, termed "sPAMC", which shows very strong specificity and high sensitivity in SARS-CoV-2 detection. However, lbCas12a does not have the ability to discriminate SNPs, nor can it be applied to one-step SNP detection.

Disclosure of Invention

Aiming at the problems that the wild LbCAs12a protein in the prior art does not have the capability of distinguishing SNP and cannot be applied to the one-step SNP detection technology, the application carries out structural modification on the wild LbCAs12a protein to obtain a mutant with the capability of distinguishing SNP, and provides a one-step SNP detection system based on the mutant.

The technical scheme provided by the application is as follows:

in a first aspect, the present application provides a mutant of a wild-type LbCas12a protein, the amino acid sequence of the wild-type LbCas12a protein being as shown in seq_1, the mutant having a mutation at least at position 174 of the amino acid sequence shown in seq_1. The 174 th position of the amino acid sequence shown in seq_1 is Arg174 (abbreviated as R in the sequence), which is located in the REC domain of Cas12a, and interacts with crRNA, arg174 mutation possibly interfering with the substrate binding ability of Cas12 a-RNP. Preferably, amino acid 174 is mutated from R to K or A.

In some embodiments provided herein, the mutant further comprises a mutation at least one of position 538, 897, 945 of the amino acid sequence shown in seq_1. The 538 st, 897 th and 945 th sites of the amino acid sequence shown in the seq_1 are Lys538, lys897 and Lys945 respectively, the Lys897 and Lys945 are both positioned in a RuvC structural domain, the Lys538 is positioned in a WED structural domain, and mutation at any one of the positions can enhance the specificity of the mutant to identify SNP. Preferably, amino acid 538 is mutated from K to R, 897 is mutated from K to A, 945 is mutated from K to A. Further preferably, the mutants are any of R174A, R174K, K538R/R174K, K897A/R174A, K945A/R174A, and the mutants have stronger SNP distinguishing ability and the enzyme activity is not affected.

In a second aspect, the present application provides a fusion protein comprising a mutant of the wild-type LbCas12a protein described above, as well as other modifications.

In a third aspect, the present application provides a polynucleotide encoding a mutant of the wild-type LbCas12a protein described above or the fusion protein described above.

In a fourth aspect, the present application provides a vector comprising a polynucleotide as described above and a regulatory element operably linked thereto.

In a fifth aspect, the present application provides an engineered host cell comprising a mutant of the wild-type LbCas12a protein described above, or the fusion protein described above, or the polynucleotide described above, or the vector described above.

In a sixth aspect, the present application provides a CRISPR-Cas system or kit for one-step detection of SNPs on double-stranded DNA, comprising a mutant of the wild-type LbCas12a protein described above or the fusion protein described above and at least one gRNA;

the gRNA includes a mutant co-repeat sequence capable of binding the wild-type LbCas12a protein described above and a guide sequence crRNA capable of targeting a target sequence.

In a seventh aspect, the present application provides a composition comprising:

(i) A protein component selected from the group consisting of: mutants of the wild type LbCas12a protein described above or the fusion proteins described above;

(ii) A nucleic acid component which is a gRNA comprising a direct repeat sequence capable of binding to a mutant of the wild-type LbCas12a protein described above and a guide sequence capable of targeting a target sequence;

the protein component and the nucleic acid component are combined with each other to form a complex.

Compared with the prior art, the application has the following beneficial effects:

compared with the wild LbCAs12a protein, the mutant has SNP identification capability, and after the CRISPR-Cas system designed based on the mutant is not classical PAM and truncates crRNA, the SNP in a genome can be accurately identified within 30 minutes, and the sample preparation process of the system is very simple, so that cross contamination can be avoided, and the operation difficulty and detection cost can be reduced.

Drawings

FIG. 1 is a schematic diagram of a SeCas12 a-based SNP detection method. In a one-step reaction, RPA and Cas12a detection are reacted simultaneously. Crrnas targeting SNPs produce high fluorescence when reacted with DNA substrates with SNPs and weak fluorescence when reacted with incompletely paired substrates.

FIG. 2 is a schematic representation of crRNA design. Wherein crRNA G/681G or crRNAA/681A refers to crRNA that is perfectly complementary to CYP2C19 x 2681G or A alleles, respectively.

Fig. 3 is a summary heat map of endpoint (60 min) fluorescent signal values generated by LbCas12a candidate mutants on CYP2C19 x 2681G and a alleles. This experiment uses crRNA G/681G to detect CYP2C19 x 2681G (targeted) and a (non-targeted) alleles.

FIG. 4 is a summary heat map of endpoint (60 minutes) fluorescence signal values generated by rescreening the candidate proteins of FIG. 3. This experiment uses crRNAA/681A to detect CYP2C19 x 2681A and G alleles. The high and low fluorescence signals are represented in red and blue, respectively.

Fig. 5 demonstrates that CrRNA truncation can further enhance SNP discrimination capability of SeCas12a, wherein:

(A-B) SeCas12a (K538R/R174K) combined with crRNA of 19nt length produced the best SNP differentiation effect. This experiment used crRNAA/42614A and crRNA C/42614C to distinguish between CYP2C9 x 342614A and C alleles.

(A) Real-time fluorescence curves for four LbCas12a RNPs. The experiment was read every 2 minutes for 1 fluorescence signal (λex=485 nm; λem=528 nm). (the dashed line is the mean ± standard deviation value, n=3).

(B) Bar graph of endpoint (30 min) fluorescence signal values generated for each LbCas12a RNP in panel (a). (n=3, mean ± standard deviation).

(C) In vitro cis-cleavage activity of each RNP in Panel (A).

(D) The cis-cleavage kinetics curves of LbCas12a and SeCas12a are shown.

(E) Shows the correlation of cis-cleavage activity with signal to noise ratio of one-step method.

Fig. 6 demonstrates the advantages of a single-step SNP detection system based on SeCas12a, wherein:

(A) Classical PAM (grey) and non-classical PAM (blue) distribution of LbCas12a in the human genome.

(B) Real-time fluorescence curves generated by non-classical PAM (5 '-TCCA-3') and classical PAM (5 '-TTTA-3') in a one-step SNP detection reaction. The experiment used crRNA A/42614A (19 nt) and crRNA C/42614C (19 nt) to detect CYP2C9 x 342614A and C alleles. (n=3, mean ± standard deviation).

(C) Schematic representation of the position of SNP sites in crRNA.

(D) Fluorescence signal of M1-M19. The position of the SNP site in the crRNA affects the specificity of the one-step SNP detection. The experiment was based on crRNAA/42614A, with sequential mutations of bases in crRNAA, so that SNPs are at different positions in the crRNA.

(E) Histogram of endpoint fluorescence values (60 th minute) in panel (D).

(F) The single-step SNP detection system is not limited to the type of base mutation. The histogram is statistical information of the endpoint fluorescence values (60 th minute). The experiment used crRNAA/42614A. The read interval of the fluorescent signal was 2 minutes (λex=485 nm; λem=528 nm). Data are mean ± standard deviation, (n=3).

Fig. 7 uses a response based on SeCas12a to detect human pharmacogenomic SNPs, wherein:

(A) CYP2C9 x 342614A and C alleles in human samples were detected using truncated crRNA. The experiment used crRNAA/42614A (19 nt) and crRNAC/42614C (19 nt), with a sample size of 3.NC is a negative control, without any DNA substrate. Data are mean ± standard deviation. (n=3).

(B) CYP2C 19X 2681G and A alleles in human samples were detected using full-length crRNA. The experiment used crRNAG/681G (20 nt) and crRNAA/681A (20 nt), with a sample size of 6.NC is a negative control, without any DNA substrate. Data are mean ± standard deviation, (n=3).

Figure 8 demonstrates the specificity and activity of Cas12a, wherein:

(A) crRNA screening results for CYP2C19 x 2681G and a alleles. The high and low fluorescence signals are displayed in red and blue, respectively.

(B) Wild type LbCas12a protein does not possess SNP discrimination capability. The experiment used full length or truncated crRNAA/42614A.

(C-D) demonstrates trans-cleavage activity of each candidate mutant of LbCAs12a in FIG. 3. The experiment used crRNAG/681G (FIG. 8C) and crRNAA/681A (FIG. 8D).

Detailed Description

The method comprises the steps of modifying wild LbCAs12a protein by taking a crystal structure as a guide, constructing a mutant series of the wild LbCAs12a protein, screening out mutant SeCas12a with better specificity and weaker activity, expanding one-step nucleic acid detection to one-step SNP detection by using the mutant, designing a simplified SNP detection system based on the SeCas12a, enabling the SNP detection and RPA amplification mediated by the Cas12a to be carried out simultaneously in the same reaction system, adding allele-specific crRNA in each independent reaction, and judging the SNP type corresponding to a substrate DNA sequence by detecting fluorescent reading of each allele-specific (figure 1). The SNP detection system uses non-classical PAM in the Cas12a system to perform ultra-rapid nucleic acid detection with higher sensitivity, reproducibility and flexibility. The sample genome of the Cas12a system has a huge number of non-classical PAM, so that the detection range of the sample genome on the genome is greatly widened. Both classical and non-classical PAM perform well in single step reactions based on SeCas12a, which allows the present application to detect SNPs at almost any location in the genome without suffering from PAM. In addition, for different targets, the detection effect can reach the optimal state by adjusting the length of crRNA. Thus, the SNP detection system of the present application can detect SNPs distributed at almost any position of the genome.

The present application is further described below in connection with the examples, which are provided solely for better illustration of the technical solution of the present application and are not intended to limit the claims. The present application is not limited to the specific examples and embodiments described herein. Further modifications and improvements may readily be made by those skilled in the art without departing from the spirit and scope of the present application, and are intended to fall within the scope of the present application.

Examples

(1) Mutant design of wild type LbCas12a protein:

the wild type LbCas12a protein is reported to be unable to recognize single nucleotide mismatches in the substrate.

Applicants speculate that the strength of binding between Cas12a-RNP and the substrate may be critical for its specificity. To verify this hypothesis, the present application mutated amino acids responsible for crRNA binding and DNA recognition, such as Ser168, arg174, asn260, ser286, lys897, ile896, gln944, lys945, phe983, lys984 and Arg883 in the helical domain, in the REC1 domain of the wild-type LbCas12a protein, to alanine a or lysine K, respectively, to alter binding affinity. The amino acids in the wild type LbCas12a protein (Asp 156, asn260, gly532 and Lys538, respectively) were also mutated in the present application, and the mutation is shown in table 1.

The amino acid sequence of the wild-type LbCas12a protein is shown as seq_1 (candidate mutation sites are respectively 156 th, 168 th, 174 th, 260 th, 286 th, 532 th, 538 rd, 883 rd, 897 th, 896 th, 944 th, 945 th, 983 rd and 984 th positions underlined):

MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH。

TABLE 1 mutation of wild type LbCAs12a protein according to the present application

(2) Design and screening of CrRNA

The design and screening of crrnas is the basis of detection reactions, and crrnas targeting two clinically relevant targets, CYP2C9 x 3 (424 a > C) and CYP2C19 x 2 (6811 g > a), were first designed and screened. And (3) comparing and analyzing the intensity of fluorescent signals generated by the allele specific crRNA and the matched genotype or the unmatched genotype, and screening crRNA with higher specific signals and lower nonspecific signals for subsequent experiments.

Table 2 42 candidate crrnas designed for CYP2C19 x 2 sites

Of the 42 candidate crrnas designed for the CYP2C19 x 2 locus, crRNA #17 and crRNA #30 had the highest specificity for their respective genotypes (fig. 8A) and were used for further optimization.

(3) Mutant screening of wild type LbCas12a protein

The present application performed a first round of screening with crRNA #17 targeting the G allele in CYP2C19 x 2 (6811G > a) for a series of mutants of wild-type LbCas12a protein constructed in table 1 (fig. 2). As shown in FIG. 3, the mutants containing the mutation site of R174A, R174K, K538R/R174K, K897A/R174A or K945A/R174A had stronger SNP distinguishing ability than the wild type LbCAs12a protein, and the enzyme activity was not affected. Of the above mutants, the specificity of R174A and R174K for recognition of SNPs was most prominent, indicating that Arg174 is associated with the specificity of LbCas12 a. Applicants believe that Arg174 is located in the REC domain of Cas12a, and that the interaction with crRNA, arg174 mutation, may interfere with the substrate binding capacity of Cas12 a-RNP. Arg174 is mutated in combination with other amino acids in the present application in order to further increase its specificity. Among them, three mutations containing two mutation sites of K538R/R174K, K897A/R174A and K945A/R174A showed higher specificity, while three mutants of K538R, K897A, K945A having only one mutation site had poor specificity, which further highlighted the importance of mutation site Arg 174. The mutant K538R/R174K containing two mutation sites is finally selected as the optimal mutant for identifying SNP, because the mutant has good stability on different targets.

(4) Mutant targeting stability screening:

to determine whether the 5 mutants screened in the first round (R174A, R174K, K538R/R174K, K897A/R174A, K945A/R174A) were stable in function when targeting the a allele in CYP2C19 x 2, a second round of screening was performed on these 5 mutants using crRNA #30 (fig. 2). The experimental results show that of these 5 mutants, only K538R/R174K showed higher specificity in targeting the a allele in CYP2C19 x 2, i.e. a very high fluorescent signal was generated in targeting the a allele in CYP2C19 x 2, whereas only a weak background fluorescence was generated in targeting the G allele (fig. 4).

(5) The effect of crRNA #17 and crRNA #30 on the enzymatic activity of the 5 mutants screened in the first round was verified:

to assess whether the enzymatic activity of the 5 mutants screened in the first round was affected, a simple trans-cleavage assay was performed in which the reaction system did not contain the RPA amplification component. In this experiment, crrna#17 and crrna#30 targeting CYP2C19 x 2 (681) G or a alleles and their corresponding substrate DNA were used in the present application (fig. 8C and 8D). Experimental results show that the enzyme activity of the 5 mutants screened in the first round is reduced to different degrees while the specificity is improved. The K538R/R174K mutant with the highest specificity has the weakest enzyme activity and stronger SNP distinguishing capability, and is named as SeCas12 (sensitive Cas12 a).

(6) The truncated crRNA further enhances the sensitivity of the SeCas12 a:

to explore whether crRNA truncation can further improve the specificity of the single-step SNP detection system based on SeCas12a, the present application uses crRNAA or crRNAC targeting the CYP2C9 x 3 (42614) a or C allele. Because of the limited number of PAMs in the CYP2C9 x 3 (424 a > C) target, only one crRNA was available, and there was no screening room, the present application performed a series of truncations of crrnas targeting this SNP site, and compared the specificity of crRNA truncations (fig. 8B). Experiments show that crRNA truncation improves crRNA specificity to some extent, but the overall discrimination effect is not ideal, so crRNA truncation alone cannot be used as a main means of discriminating SNPs.

Table 3 candidate crrnas designed for CYP2C9 x 3 in the present application

By comparing the endpoint fluorescence signal values by analysis, it was found that only crRNAC of length 18nt, when used in combination with SeCas12a, did not generate a sufficiently strong fluorescence signal, and crRNAC of length 19nt, when used in combination with SeCas12a, had the highest specificity of the SeCas12a detection system (fig. 5A). In contrast, the CRISPR-Cas system, with a length of 19nt crRNA C in combination with the wild-type LbCas12a protein, was able to target the a allele but produced a large amount of non-specific fluorescent signal (fig. 5A). The present application also found that crRNAA shows high specificity at all three lengths (fig. 5B).

(7) One-step SNP detection system design based on SeCas12a

Studies have shown that dynamic balance between CRISPR activity and RPA amplification is critical for one-step detection reactions. In one aspect, CRISPR detection requires amplification of large amounts of target DNA by RPA to generate a sufficiently strong fluorescent signal; on the other hand, CRISPR detection systems require cleavage of target DNA to generate a fluorescent signal. To achieve this equilibrium, it is necessary to down-regulate CRISPR kinetics in order to achieve RPA-mediated mass amplification of target sequences at the early stages of the one-step detection reaction. Whereas in SNP detection, the targeted and non-targeted DNA sequences differ only by one nucleotide, so a more stringent balance of CRISPR dynamics between targeted and non-targeted is required.

The cis-cleavage activity of SeCas12a was quantified with crRNAA and crRNA C (FIG. 5C). crRNAA exhibits weaker cis-cleavage activity when used in combination with wild-type Cas12a protein or SeCas12 a; whereas crRNAC exhibits higher cis-cleavage activity when paired with SeCas12a (fig. 5C). Furthermore, crrnas of 18nt length exhibited very weak or even no cis-cleavage activity when used in combination with wild-type Cas12a protein or SeCas12a (fig. 5C). This may explain why crrnas of 18nt length do not produce sufficient fluorescent signals. These data indicate that only when cis-cleavage activity is low to a certain threshold, it specifically cleaves perfectly matched substrates and not incompletely matched substrates, thereby selectively generating a fluorescent signal.

Taken together, crRNA length is critical in single-step SNP detection based on SeCas12 a. For target sites exhibiting strong cis-cleavage activity, a truncated crRNA (19 nt) is required, whereas target sites exhibiting weak cis-cleavage activity require crrnas of 20nt in length.

(8) Single-step SNP detection system based on SeCas12a

The present application contemplates a single step SNP detection system based on SeCas12a in which the present application uses non-classical PAM instead of classical PAM. Thus, the one-step SNP detection system can be used as a generalized platform for detecting almost any target in the genome. Statistical data indicate that the number of non-classical PAMs of LbCas12a in the human genome is 4.62 times that of classical PAM (fig. 6A).

To verify whether classical PAM is suitable for use in a single-step SNP detection system based on SeCas12a, the present application mutates non-classical PAM (TCCA) of crRNAA or crRNA C targeting the CYP2C9 x 3 (42614 as shown in seq_50) a or C allele to classical PAM (TTTA) as a substrate for the single-step SNP detection system. The amino acid sequence of seq_50 is:

tgtgcatctgtaaccatcctctctttaagtttgcatatacttccagcactataatttaaatttataatgatgtttggataccttcatgattcatatacccctgaattgctacaacaaatgtgccatttttctccttttccatcagtttttacttgtgtcttatcagctaaagtccaggaagagattgaacgtgtgattggcagaaaccggagcccctgcatgcaagacaggagccacatgccctacacagatgctgtggtgcacgaggTCCAgagatacattgaccttctccccaccagcctgccccatgcagtgacctgtgacattaaattcagaaactatctcattcccaaggtaagtttgtttctcctacactgcaactccatgttttcgaagtccccaaattcatagtatcatttttaaacctctaccatcaccgggtgagagaagtgcataactcatatgtatggcagtttaactggactttctcttgtttggtacctcgcgaatgcatctagatatcggatcccgggcccgtcgactgcagaggcctgcatgcaagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagcta, note: uppercase TCCA is a non-classical PAM, underlined is the spacer region.

It was found that 19nt CrRNAA showed similar specificity on substrates containing classical or non-classical PAM, whereas 19nt crRNAC produced a background signal of a certain intensity when targeting substrates containing classical PAM (fig. 6B), indicating that PAM selection had to be tested and screened separately for each crRNA to meet its kinetic requirements.

Next, the present application determines whether the position of a SNP within a spacer affects the detection effect of a one-step detection reaction by sequentially single mutating the nucleotides within the spacer region on the substrate DNA. Wherein M0 represents no mutation, and M1 to M19 represent mutations at the specified positions (FIG. 6C). As shown in FIG. 6D, in addition to M18 and M19 producing high fluorescence signals, M1 through M7 and M14-M17 each produce weak fluorescence signals distinguishable from the wild type substrate (M0), while M8 through M13 produce background level signals. In summary, the one-step response based on SeCas12a can distinguish mutations in the PAM proximal region from the middle spacer, but not in the PAM distal region. In short, in the design of crrnas, a distinct distinction of specific fluorescent signals from background signals can be achieved as long as SNPs are in the 1-13 position (from the PAM side). As shown in table 4:

TABLE 4 mutation site of M0-M19 crRNA sequence (19 nt)

Note that: underlined are mutation sites.

To explore whether a single-step SNP detection system based on SeCas12a was able to detect base transitions and base transversions, the present application mutated the CYP2C9 x 3 (42614) a allele to C, G and T, respectively, and conducted SNP detection experiments. The results show that both crRNAA and crRNAC can effectively distinguish base transitions or base transversions (fig. 6E). Quantitative analysis of endpoint fluorescence signals also demonstrated that crRNAA produced significantly higher fluorescence signals on specific targets (a) than on C, G and T targets. Likewise, crRNAC produced a very high fluorescent signal at specific target (C), while only background level fluorescent signal was produced at A, G and T targets (fig. 6E). In summary, the single-step SNP detection system based on SeCas12a can utilize both classical and non-classical PAM and is hardly limited by mutation positions and mutation types.

(9) Detection of pharmacogenomic SNPs using a single-step SNP detection system based on SeCas12a

CYP2C9 x 3 (424A > C) and CYP2C19 x 2 (6811G > A) in a human sample are detected by the one-step SNP detection system based on the SeCas12 a. Crrnas a and C of length 19nt were used to detect CYP2C9 x 342614a and C alleles and crrnas #17 and #30 were used to detect CYP2C19 x 2681G and a alleles.

TABLE 5 RPA primers used in experiments

The experimental procedure was as follows:

s1, preparing a sample: human oral epithelial cells were gently scraped off into 500. Mu.L of PBS buffer and centrifuged at 300rcf for 5 min (10 min at 200 g). The supernatant was discarded, and 100ul of PBS (400. Mu.L of supernatant discarded) and proteinase K (final concentration 200. Mu.g/ml) were added. Digestion at 55℃for 60 min and heating at 80℃for 10min deactivates proteinase K.

S2, preparing a reaction system.

Adding 30 mu LABuffer,10 mu L of non-ribozyme water, 4 mu L of forward RPA primer and 4 mu L of reverse RPA primer into each tube of RPA powder (Anpu future), shaking, mixing, standing at room temperature for 2min to dissolve the powder; adding 9 mu L of dissolved RPA reagent, 3 mu L of seCas12a (1 mu M), 3 mu L of crRNA (1 mu M), 4 mu L of ssDNAreport (4 mu M,5 '-TTATT-3'), shaking for 10s, mixing uniformly, and standing at room temperature for 10min to incubate to form RNP; sequentially adding 2 mu L of samples and 2.5 mu LB buffer, rapidly sealing, and immediately placing in an enzyme-labeled instrument for detection.

S3, detecting fluorescence by using an enzyme-labeled instrument. Fluorescence values (excitation light 485nm, emission light 528 nm) were read every minute at 37℃to generate a kinetic profile.

In the above experiments, the oral epithelial cells of the volunteers were rapidly dissolved into buffer solution and used as detection templates directly without purification. The corresponding SNP was accurately detected by the SeCas12 detection system within 30 minutes in 3 samples corresponding to the CYP2C9 x 3 target (fig. 7A). In 6 samples of the present application using crRNA #17 and crRNA #30 to detect CYP2C19 x 2681G and a alleles, crRNA #17 and crRNA #30 each successfully produced allele-specific fluorescent signals within 30 minutes (fig. 7B). In summary, the one-step SNP detection system based on SeCas12a can accurately detect two pharmacogenomic SNPs in a crude solution of a human sample.

While the preferred embodiments of the present application have been described, it is of course not intended to limit the scope of the claims, and it should be noted that modifications and variations could be made by persons skilled in the art without departing from the principles of the present application, and these modifications and variations are also considered to be within the scope of the present application.

Claims

1. A mutant of a wild-type LbCas12a protein, the amino acid sequence of which is shown in seq_1, characterized in that: the mutant is any one of R174A, R, 174K, K538R and R174K, K897A and R174A, K945A and R174A, and the mutant is a mutation based on the amino acid sequence shown in seq_1.

2. A polynucleotide, characterized in that: a mutant encoding the wild-type LbCas12a protein of claim 1.

3. A vector comprising the polynucleotide of claim 2 and operably linked thereto a regulatory element.

4. An engineered host cell comprising a mutant of the wild-type LbCas12a protein of claim 1, or the polynucleotide of claim 2, or the vector of claim 3.

5. A CRISPR-Cas system or kit for one-step detection of SNPs on double-stranded DNA, characterized in that the CRISPR-Cas system or kit comprises a mutant of the wild-type LbCas12a protein of claim 1 and at least one gRNA;

the gRNA comprises a cognate repeat sequence capable of binding to a mutant of the wild-type LbCas12a protein of claim 1 and a guide sequence crRNA capable of targeting a target sequence.

6. The CRISPR-Cas system or kit for one-step detection of SNPs on double-stranded DNA according to claim 5, characterized by: the crRNA has a sequence length of 19nt.

7. The CRISPR-Cas system or kit for one-step detection of SNPs on double-stranded DNA according to claim 5, characterized in that: the SNPs are located 1 st to 13 th nt on the double-stranded DNA from the PAM side.

8. A composition, characterized in that it comprises:

(i) A protein component selected from the group consisting of: a mutant of the wild-type LbCas12a protein of claim 1;

(ii) A nucleic acid component that is a gRNA comprising a direct repeat sequence capable of binding to a mutant of the wild-type LbCas12a protein of claim 1 and a guide sequence capable of targeting a target sequence;

the protein component and the nucleic acid component are bound to each other to form a complex.