CN116286734B - Mutants of wild-type LbCas12a protein and their uses for SNP detection - Google Patents

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

Mutants of wild-type LbCas12a protein and their uses for SNP detection

Technical field

This application belongs to the biological field, and specifically relates to a mutant of wild-type LbCas12a protein and its SNP detection use.

Background technique

Single nucleotide polymorphism (SNP) mainly includes single base conversion, base transversion, base deletion or base insertion. It is the most common type of heritable variation in the human genome, with an average of 1-1.9kb. There is a SNP. SNPs are involved in many biological processes, including genetic diseases, tumorigenesis, drug metabolism, etc. Pharmacogenomic SNPs can be divided into four major categories according to their functions: SNPs related to drug absorption, drug transport, drug metabolism and drug excretion, which are closely related to individual sensitivity to drug treatment. The cytochrome P450 (CYP) family is the most important drug metabolizing enzyme, mediating the metabolism of various drugs. There are more than 2,000 CYP single nucleotide polymorphisms reported so far, and some of these mutations have a significant impact on its enzyme 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 precise personalized prescription to balance between over-anticoagulation (blood clots) and under-anticoagulation (bleeding). Achieve precise balance. Studies have found that two common variants of CYP2C9, namely CYP2C9*2(3608C>T) and CYP2C9*3(42614A>C), lead to reduced enzyme activity. Heterozygous or homozygous individuals carrying the above mutations are more sensitive to warfarin than normal individuals. Therefore, they face a greater risk of bleeding during warfarin treatment and require lower doses. For another example, CYP2C19 is the key metabolic enzyme of clopidogrel. Clopidogrel is an effective anti-platelet aggregation drug. It is metabolized by CYP2C19 to produce 2-oxo-clopidogrel, and is eventually metabolized into an active thiol metabolite, which exerts the function of inhibiting platelet aggregation. For patients who carry the CYP2C19*2 (681G>A) allele in their genome, due to the loss of CYP2C19 enzyme activity in their bodies, treatment with clopidogrel will result in ineffective treatment, delayed disease progression, and risk of bleeding. Therefore, rapid and accurate detection of key pharmacogenomic SNPs is crucial for personalized medicine when using high-risk drugs. Since CYP2C9 and CYP2C19 play important functions in the metabolism of warfarin, clopidogrel, phenytoin, siponimod, valproate and other drugs, a rapid test was developed using CYP2C9 and CYP2C19 as detection targets. Accurate detection methods are very important.

At present, a variety of SNP detection methods have been developed, which can be roughly divided into two categories: electrophoresis-based methods and high-throughput identification methods. SNP detection based on electrophoresis, such as denaturing gradient gel electrophoresis, allele-specific PCR, and cleavage-amplified polymorphic sequence tags, all have the characteristics of being time-consuming, low in accuracy, and capable of identifying but unable to identify 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 fields such as genome editing and nucleic acid detection. Among them, V-type and VI-type Cas proteins, such as Cas12a, Cas12b, Cas13a and Cas14, can initiate non-specific trans-cleavage when specific cis-cleavage is generated. This property is the basis of CRISPR detection systems. These Cas proteins can recognize and cut target DNA sequences under the guidance of crRNA, and then generate non-specific trans-cleaving activity to start cutting single-stranded DNA or RNA. Taking advantage of the above characteristics, the detection signal amplification can be achieved by adding reporter DNA or RNA sequences to the detection system. Among the above-mentioned Cas proteins, Cas13a and Cas14 can distinguish SNPs in RNA or single-stranded DNA substrates respectively. However, since the amplification of RNA substrates and single-stranded DNA substrates requires reverse transcription or single-stranded DNA generation steps, it cannot be applied to a one-step detection system; introducing mutations in the crRNA of Cas12b can distinguish double-stranded DNA substrates. SNP in , but its optimal cutting temperature is 48°C, which greatly limits its compatibility with isothermal amplification technology. Since the optimal reaction temperature of recombinase polymerase amplification (RPA) is 37-42°C, Cas12b must be used in conjunction with loop-mediated isothermal amplification (LAMP), and the optimal reaction temperature of LAMP is 55-65°C. , changes in reaction temperature will reduce the cleavage activity of Cas12b and extend the incubation time, ultimately leading to a slowdown in detection speed.

In contrast, Cas12a is a perfect match for RPA. Cas12a uses double-stranded DNA as a substrate and is widely used in nucleic acid detection. However, the need for TTTV-PAM and low specificity limit its application in SNP detection. Even if crRNA is extended, including adding an additional TA-rich DNA sequence to the 3' end of crRNA and inserting nucleotides in the spacer (nucleotides at the 3' end of crRNA and the spacer), SNPs in the substrate cannot be distinguished well. By adding a classic PAM sequence upstream of the target site during PCR amplification and introducing additional mismatches on crRNA, Cas12a can distinguish SNPs in a two-step reaction. Recent studies have disclosed a one-step nucleic acid detection system based on LbCas12a, named "sPAMC", which showed strong specificity and high sensitivity in SARS-CoV-2 detection. However, LbCas12a does not have the ability to distinguish SNPs and cannot be used in one-step SNP detection.

Contents of the invention

In order to solve the problem in the existing technology that the wild-type LbCas12a protein does not have the ability to distinguish SNPs and cannot be used in one-step SNP detection technology, this application structurally modified the wild-type LbCas12a protein to obtain a mutant with the ability to distinguish SNPs. , and provide a one-step SNP detection system based on this mutant.

The technical solutions provided by this application are as follows:

In the first aspect, this application provides a mutant of the wild-type LbCas12a protein. The amino acid sequence of the wild-type LbCas12a protein is shown in Seq_1, and the mutant has a mutation at least at position 174 of the amino acid sequence shown in Seq_1. Position 174 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. The Arg174 mutation may interfere with the substrate binding ability of Cas12a-RNP. Preferably, the amino acid at position 174 is mutated from R to K or A.

In some embodiments provided herein, the mutant also has a mutation in at least one of the 538th, 897th, and 945th positions of the amino acid sequence shown in Seq_1. The 538th, 897th, and 945th positions of the amino acid sequence shown in Seq_1 are Lys538, Lys897, and Lys945, respectively. Lys897 and Lys945 are both located in the RuvC domain, and Lys538 is located in the WED domain. Mutations in any of these positions can enhance the specificity of the mutant in recognizing SNPs. Preferably, the amino acid at position 538 mutates from K to R, the amino acid at position 897 mutates from K to A, and the amino acid at position 945 mutates from K to A. Further preferably, the mutant is any one of R174A, R174K, K538R/R174K, K897A/R174A, and K945A/R174A, which have stronger SNP differentiation capabilities and their enzyme activities are not affected.

In a second aspect, the present application provides a fusion protein that includes a mutant of the above wild-type LbCas12a protein and other modified parts.

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

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

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

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

The above-mentioned gRNA includes a direct repeat sequence of a mutant capable of binding to the above-mentioned wild-type LbCas12a protein and a guide sequence crRNA capable of targeting a target sequence.

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

(i) Protein component, which is selected from: a mutant of the above wild-type LbCas12a protein or the above fusion protein;

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

The above-mentioned protein component and the above-mentioned nucleic acid component combine with each other to form a complex.

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

The present application provides a mutant of a wild-type LbCas12a protein, which has SNP recognition ability compared with the wild-type LbCas12a protein. The CRISPR-Cas system designed based on the mutant can accurately identify SNPs in the genome within 30 minutes after non-classical PAM and truncated crRNA. The sample preparation process of the system is very simple, which can avoid cross-contamination and reduce the operation difficulty and detection cost.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the SNP detection method based on SeCas12a. In a one-step reaction, RPA and Cas12a detection react simultaneously. SNP-targeting crRNA produces high fluorescence when reacting with DNA substrates containing SNP, and weak fluorescence when reacting with incompletely paired substrates.

Figure 2 is a schematic diagram of crRNA design. Among them, crRNA G/681G or crRNAA/681A refers to crRNA that is completely complementary to CYP2C19*2681G or A allele respectively.

Figure 3 is a heat map summarizing the end-point (60th minute) fluorescence signal values produced by LbCas12a candidate mutants on CYP2C19*2681G and A alleles. This experiment uses crRNA G/681G to detect CYP2C19*2681G (targeting) and A (non-targeting) alleles.

Figure 4 is a heat map summarizing the endpoint (60th minute) fluorescence signal values generated by re-screening the candidate proteins in Figure 3. This experiment uses crRNAA/681A to detect CYP2C19*2681A and G alleles. High and low fluorescence signals are shown in red and blue, respectively.

Figure 5 shows that CrRNA truncation can further improve the SNP discrimination ability of SeCas12a, where:

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

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

(B) Bar chart of end-point (30 minutes) fluorescence signal values generated by each LbCas12a RNP in panel (A). (n=3, mean ± standard deviation).

(C) In vitro cis-cleaving activity of each RNP in panel (A).

(D) shows the cis-cleavage kinetic curves of LbCas12a and SeCas12a.

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

FIG6 shows the advantages of the one-step SNP detection system based on SeCas12a, where:

(A) Distribution of classical PAM (gray) and non-classical PAM (blue) of LbCas12a in the human genome.

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

(C) Schematic diagram of the location of the SNP site in crRNA.

(D) Fluorescence signal of M1-M19. The location of the SNP site in crRNA will affect the specificity of one-step SNP detection. This experiment is based on crRNAA/42614A and sequentially mutates the bases in crRNAA so that the SNP is at different positions of crRNA.

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

(F) The one-step SNP detection system is not limited to base mutation types. The histogram is the statistical information of the end-point fluorescence value (at 60 minutes). This experiment used crRNAA/42614A. The fluorescence signal was read at an interval of 2 minutes (λex=485nm; λem=528nm). Data are means ± standard deviation, (n=3).

Figure 7 Detection of human pharmacogenomic SNPs using SeCas12a-based reactions, where:

(A) Detection of CYP2C9*342614A and C alleles in human samples using truncated crRNA. This experiment used crRNAA/42614A (19nt) and crRNAC/42614C (19nt) with a sample size of 3. NC is a negative control and does not contain any DNA substrate. Data are means ± standard deviation. (n=3).

(B) Detection of CYP2C19*2681G and A alleles in human samples using full-length crRNA. This 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 ± SD, (n = 3).

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

(A) Results of crRNA screening for CYP2C19*2681G and A alleles. High and low fluorescence signals are shown in red and blue, respectively.

(B) Wild-type LbCas12a protein does not have the ability to distinguish SNPs. This experiment used full-length or truncated crRNAA/42614A.

(C-D) shows the trans-cleaving activity of each candidate mutant of LbCas12a in Figure 3. This experiment used crRNAG/681G (Fig. 8C) and crRNAA/681A (Fig. 8D).

Detailed ways

This application uses the crystal structure as a guide to transform the wild-type LbCas12a protein, construct a mutant series of the wild-type LbCas12a protein, and screen out the mutant SeCas12a with better specificity and weaker activity, which has the best SNP distinguishing ability. Strong, this application uses mutants to expand one-step nucleic acid detection to one-step SNP detection. A simplified SNP detection system is designed based on SeCas12a, so that Cas12a-mediated SNP detection and RPA amplification can be performed simultaneously in the same reaction system, and Allele-specific crRNA is added to each independent reaction, and by detecting the fluorescence reading specific to each allele, the SNP type corresponding to the substrate DNA sequence can be determined (Figure 1). This SNP detection system uses non-classical PAM in the Cas12a system to perform ultra-fast nucleic acid detection with higher sensitivity, reproducibility and flexibility. There are a large number of non-classical PAMs in the genome of Cas12a system samples, which greatly broadens its detection range on the genome. Both classical PAM and non-classical PAM perform well in the one-step reaction based on SeCas12a, which allows this application to detect SNPs at almost any position in the genome without being troubled by PAM. In addition, for different targets, this application can adjust the length of crRNA to achieve the best detection effect. Therefore, the SNP detection system of the present application can detect SNPs distributed at almost any position in the genome.

The present application is further described below in conjunction with the examples, which are only intended to better illustrate the technical solution of the present application rather than to limit the claims. The present application is not limited to the specific embodiments and implementation schemes described herein. Any technician in the art can easily make further improvements and perfections without departing from the spirit and scope of the present application, which fall within the scope of protection of the present application.

Example

(1) Design of mutants of wild-type LbCas12a protein:

It has been reported that the wild-type LbCas12a protein cannot recognize single nucleotide mismatches in the substrate.

Applicants speculate that the binding strength between Cas12a-RNP and substrate may be critical to its specificity. To test this hypothesis, this application will identify the amino acids responsible for crRNA binding and DNA recognition, such as Ser168, Arg174, Asn260, Ser286, Lys897, Ile896, Gln944, Lys945, Phe983, Lys984 and the helical structure in the REC1 domain of the wild-type LbCas12a protein. Arg883 in the domain, was mutated to alanine A or lysine K respectively to change the binding affinity. In this application, the amino acids in the wild-type LbCas12a protein (Asp156, Asn260, Gly532 and Lys538 respectively) were also mutated. The mutation status is shown in Table 1.

The amino acid sequence of the wild-type LbCas12a protein is shown in Seq_1 (the candidate mutation sites are underlined, which are positions 156, 168, 174, 260, 286, 532, 538, and No. 883, 897, 896, 944, 945, 983, 984):

MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFF D NRENMFSEEAK S TSIAF R CINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYI N LYNQKTKQKLPKFKPLYKQVLSDRE S LSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYD KVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFM G GWKD K ETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLS GGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKE R FEARQNWTSIEN IK ELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVY QK FEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFES FK SMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDE KLDKVKIAISNKEWLEYAQTSVKH.

Table 1 The mutation status of wild-type LbCas12a protein in this application

(2)Design and screening of CrRNA

The design and screening of crRNA is the basis of the detection reaction. This application first designed and screened crRNAs targeting two clinically relevant targets, CYP2C9*3 (42614A>C) and CYP2C19*2 (681G>A). By comparing and analyzing the intensity of the fluorescence signal generated by the allele-specific crRNA and its matching genotype or mismatched genotype, the crRNA with higher specific signal and lower nonspecific signal was screened for subsequent experiments.

Table 2 42 candidate crRNAs designed for the CYP2C19*2 locus

Among the 42 candidate crRNAs designed for the CYP2C19*2 locus, crRNA#17 and crRNA#30 had the highest specificity for their respective genotypes ( Figure 8A ) and were used for further optimization.

(3) Screening of mutants of wild-type LbCas12a protein

The present application uses crRNA#17 targeting the G allele in CYP2C19*2 (681G>A) to screen the mutants of a series of wild-type LbCas12a proteins constructed in Table 1 in the first round (Fig. 2). As shown in Figure 3, compared with wild-type LbCas12a protein, mutants containing R174A, R174K, K538R/R174K, K897A/R174A or K945A/R174A mutation sites have stronger SNP differentiation ability, and their enzyme activity is not affected. In the above mutants, the specificity of R174A and R174K recognition of SNP is the most prominent, which shows that Arg174 is related to the specificity of LbCas12a. The applicant believes that Arg174 is located in the REC domain of Cas12a, interacts with crRNA, and the mutation of Arg174 may interfere with the substrate binding ability of Cas12a-RNP. The present application mutates Arg174 in combination with other amino acids in order to further improve its specificity. Among them, three mutants containing two mutation sites, K538R/R174K, K897A/R174A and K945A/R174A, showed high specificity, while the three mutants K538R, K897A and K945A, which had only one mutation site, had poor specificity, which further highlighted the importance of the mutation site Arg174. The present application finally selected the mutant K538R/R174K containing two mutation sites as the best mutant for identifying SNPs because it has good stability at different target sites.

(4) Targeted stability screening of mutants:

In order to determine whether the 5 mutants (R174A, R174K, K538R/R174K, K897A/R174A, K945A/R174A) screened in the first round are functionally stable when targeting the A allele in CYP2C19*2, the present application used crRNA#30 to perform a second round of screening on these 5 mutants (Figure 2). The experimental results showed that among these 5 mutants, only K538R/R174K showed high specificity when targeting the A allele in CYP2C19*2, that is, it produced a high fluorescence signal when targeting the A allele in CYP2C19*2, and only produced weak background fluorescence when targeting the G allele (Figure 4).

(5) Verify the impact of crRNA#17 and crRNA#30 on the enzyme activity of the five mutants selected in the first round:

In order to evaluate whether the enzyme activity of the five mutants screened in the first round was affected, the present application conducted a simple trans-cleavage experiment, and the RPA amplification component was not included in the reaction system. In this experiment, the present application used crRNA#17 and crRNA#30 targeting CYP2C19*2(681)G or A alleles and their corresponding substrate DNA (Figures 8C and 8D). The experimental results showed that the enzyme activity of the five mutants screened in the first round decreased to varying degrees while the specificity was improved. Among them, the K538R/R174K mutant with the highest specificity has the weakest enzyme activity and has a stronger SNP differentiation ability, and is named SeCas12 (sensitive Cas12a).

(6) Truncated crRNA further enhances the sensitivity of SeCas12a:

To explore whether crRNA truncation can further improve the specificity of the one-step SNP detection system based on SeCas12a, this application used crRNAA or crRNAC targeting the A or C allele of CYP2C9*3(42614). Since the number of PAMs in the CYP2C9*3 (42614A>C) target site is limited, only one crRNA is available, and there is no room for screening. This application conducted a series of truncation of the crRNA targeting this SNP site, and compared the crRNA truncation body specificity (Figure 8B). Experiments show that crRNA truncation improves the specificity of crRNA to a certain extent, but the overall discrimination effect is not ideal. Therefore, crRNA truncation alone cannot be used as the main means to distinguish SNPs.

Table 3 Candidate crRNA designed in this application for CYP2C9*3

By analyzing and comparing the end-point fluorescence signal values, it was found that only crRNAC with a length of 18 nt cannot produce a strong enough fluorescence signal when combined with SeCas12a. When crRNAC with a length of 19 nt is combined with SeCas12a, the SeCas12a detection system has the highest specificity (Figure 5A). In contrast, the CRISPR-Cas system combining crRNA C with a length of 19 nt and wild-type LbCas12a protein can target the A allele, but generates a large amount of non-specific fluorescence signal (Figure 5A). The present application also found that crRNAA showed high specificity at all three lengths (Figure 5B).

(7) Design of a one-step SNP detection system based on SeCas12a

Research shows that the dynamic balance between CRISPR activity and RPA amplification is key to the one-step detection reaction. On the one hand, CRISPR detection requires the amplification of a large amount of target DNA through RPA to generate a strong enough fluorescence signal; on the other hand, the CRISPR detection system needs to cleave the target DNA to generate a fluorescence signal. To achieve the above balance, CRISPR kinetics need to be downregulated to achieve RPA-mediated massive amplification of the target sequence in the early stages of the one-step detection reaction. In SNP detection, there is only one nucleotide difference between the targeted and non-targeted DNA sequences, so a more stringent balance of CRISPR dynamics between targeting and non-targeting is required.

This application quantified the cis-cleaving activity of SeCas12a using crRNAA and crRNA C (Figure 5C). When combined with wild-type Cas12a protein or SeCas12a, crRNAA showed weak cis-cleaving activity; while when paired with SeCas12a, crRNAC showed higher cis-cleaving activity (Figure 5C). In addition, crRNA with a length of 18 nt exhibited very weak or even no cis-cleaving activity when combined with wild-type Cas12a protein or SeCas12a (Figure 5C). This may explain why crRNA with a length of 18 nt cannot produce sufficient fluorescence signal. These data indicate that only when the cis-cleaving activity is low to a certain threshold, it can specifically cleave perfectly paired substrates but not imperfectly paired substrates, thereby selectively generating a fluorescent signal.

In summary, crRNA length is crucial in one-step SNP detection based on SeCas12a. For target sites showing strong cis-cleaving activity, a truncated crRNA (19nt) is required, while for target sites showing weak cis-cleaving activity, a crRNA with a length of 20nt is required.

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

This application designs a one-step SNP detection system based on SeCas12a. In this system, this application uses non-classical PAM instead of classic PAM. Therefore, this one-step SNP detection system can be used as a universal platform to detect almost any target in the genome. Statistics show that the number of non-canonical PAMs of LbCas12a in the human genome is 4.62 times higher than that of canonical PAMs (Figure 6A).

In order to verify whether classical PAM is suitable for the one-step SNP detection system based on SeCas12a, this application will target non-classical PAM (TCCA) mutations of crRNAA or crRNA C of CYP2C9*3 (42614 as shown in Seq_50) A or C allele It is the classic PAM (TTTA) and serves as the substrate for the one-step SNP detection system. The amino acid sequence of Seq_50 is:

tgtgcatctgtaaccatcctctctttaagtttgcatatacttccagcactataatttaaatttataatgatgtttggataccttcatgattcatatacccctgaattgctacaacaaatgtgccatttttctccttttccatcagtttttacttgtgtcttatcagctaaagtccaggaagagattgaacgtgtgattggcagaaac cggagcccctgcatgcaagacaggagccacatgccctacacagatgctgtggtgcacgaggTCCA gagatacattgaccttctc cccaccagcctgccccatgcagtgacctgtgacattaaattcagaaactatctcattcccaaggtaagtttgtttctcctacactgcaactccatgttttcgaagtccccaaattcatagtatcatttttaaacctctaccatcaccgggtgagagaagtgcataactcatatgtatggcagtttaactggactttctcttgtttggtacctc gcgaatgcatctagatatcggatcccgggcccgtcgactgcagaggcctgcatgcaagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaaagcctggggtgcctaatgagtgagcta, Note: The uppercase TCCA is non-classic PAM, underlined is the spacer area.

The study found that 19nt crRNAA showed similar specificity on substrates containing canonical or non-canonical PAMs, while 19nt crRNAC produced a certain intensity of background signal when targeting substrates containing canonical PAMs (Figure 6B), indicating that PAM selection must be tested and screened individually for each crRNA to meet its kinetic requirements.

Next, this application determines whether the position of the SNP in the spacer will affect the detection effect of the one-step detection reaction by sequentially mutating the nucleotides in the spacer region on the substrate DNA. Among them, M0 indicates no mutation, and M1 to M19 indicate mutations at the specified positions (Figure 6C). As shown in Figure 6D , in addition to M18 and M19 producing high fluorescence signals, M1 to M7 and M14-M17 all produced weak fluorescence signals that could be distinguished from the wild-type substrate (M0), while M8 to M13 produced background-level signals. In summary, the one-step reaction based on SeCas12a can distinguish mutations in the proximal region of PAM and the middle spacer region, but cannot distinguish mutations in the distal region of PAM. In short, when designing crRNA, as long as the SNP is at positions 1-13 (from the PAM side), the specific fluorescence signal can be clearly distinguished from the background signal. As shown in Table 4:

Table 4 Mutation sites of M0-M19 crRNA sequence (19nt)

Note: Underlined are mutation sites.

In order to explore whether the one-step SNP detection system based on SeCas12a can detect base conversion and base transversion, this application mutated the CYP2C9*3 (42614) A allele to C, G and T respectively to conduct SNP detection experiments. The results showed that both crRNAA and crRNAC could effectively distinguish base transitions or base transversions (Figure 6E). Quantitative analysis of end-point fluorescence signals also confirmed that crRNAA produced significantly higher fluorescence signals at the specific target site (A) than at the C, G, and T target sites. Similarly, crRNAC produced a high fluorescence signal on the specific target site (C), while only producing background-level fluorescence signals on the A, G, and T target sites (Figure 6E). In summary, the one-step SNP detection system based on SeCas12a can utilize both classical and non-classical PAMs and is almost unrestricted by mutation location and mutation type.

(9) Detecting pharmacogenomic SNPs using a one-step SNP detection system based on SeCas12a

This application uses a one-step SNP detection system based on SeCas12a to detect CYP2C9*3 (42614A>C) and CYP2C19*2 (681G>A) in human samples. This application uses crRNAA and crRNA C with a length of 19nt to detect CYP2C9*342614A and C alleles, and crRNA#17 and crRNA#30 to detect CYP2C19*2681G and A alleles.

Table 5 RPA primers used in experiments

The experimental steps are as follows:

S1. Sample preparation: Gently scrape human oral epithelial cells into 500 μL PBS buffer, centrifuge at 300 rcf for 5 minutes (200 g for 10 minutes). Discard the supernatant, add 100 ul PBS (discard 400 μL supernatant) and proteinase K (final concentration 200 μg/ml). Digest at 55°C for 60 minutes, and heat at 80°C for 10 minutes to inactivate proteinase K.

S2. Prepare the reaction system.

Add 30 μL of LABuffer, 10 μL of ribozyme-free water, 4 μL of forward RPA primer, and 4 μL of reverse RPA primer to each tube of RPA powder (Amp Future). Shake and mix and place at room temperature for 2 minutes to dissolve the powder. Add 9 μL of each detection system to dissolve. Good RPA reagent, 3μL seCas12a (1μM), 3μL crRNA (1μM), 4μL ssDNAreport (4μM, 5'-TTATT-3'), shake for 10s to mix, let stand at room temperature for 10min to incubate to form RNP; add 2μL of sample in sequence, 2.5 μLB buffer, seal it quickly and immediately place it in a microplate reader for detection.

S3. Use a microplate reader to detect fluorescence. The fluorescence value (excitation light 485nm, emission light 528nm) was read every minute at 37°C to generate a kinetic curve.

In the above experiments, volunteers' oral epithelial cells were quickly dissolved into buffer and could be directly used as detection templates without purification. In the three samples corresponding to the CYP2C9*3 target, the SeCas12 detection system accurately detected the corresponding SNP within 30 minutes (Figure 7A). In this application, crRNA#17 and crRNA#30 were used to detect the 6 samples of CYP2C19*2681G and A alleles. Both crRNA#17 and crRNA#30 successfully generated allele-specific fluorescence signals within 30 minutes (Figure 7B ). In summary, the one-step SNP detection system based on SeCas12a can accurately detect two pharmacogenomic SNPs in crude lysates of human samples.

The above is only the preferred implementation mode of the present application, which certainly cannot be used to limit the scope of rights of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and changes can be made without departing from the principles of the present application, and these improvements and changes are also regarded as the scope of protection of the present application.

Claims (8)

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.