Cas12a homologue pscas12a and mutant thereof, and use thereof in gene editing

Abstract

Provided are a Cas12a homologue PsCas12a and a mutant thereof, and the use thereof in gene editing. Provided is a Cas12a homologue PsCas12a, having an amino acid sequence as shown in SEQ ID NO: 1. Compared with Cas12a, PsCas12a has a better editing effect in terms of gene editing efficiency, PAM compatibility, specificity, etc. By means of sequence screening and design optimization, a series of mutants of PsCas12a having higher editing efficiency and further expanded PAM compatibility are obtained. By means of in-vitro and in-vivo cleavage experiments, these mutants have better gene editing effects in genome editing. The provided PsCas12a and mutants thereof have broad application prospects in the genome editing of plants, animals or microorganisms.

Description

PsCas12a, a homolog of Cas12a, its mutants, and their applications in gene editing. Technical Field

[0001] This invention relates to Cas12a homologs and their applications, particularly to the Cas12a homolog PsCas12a and its mutants and their applications in genome editing, belonging to the field of Cas12a homologs and their applications in genome editing. Background Technology

[0002] In recent years, gene editing technology has developed rapidly. Among them, the CRISPR-Cas system has become an important tool in biomedical research and genetic engineering due to its high efficiency, specificity, and ease of operation. Cas12a is a single effector protein in the CRISPR family, showing unique advantages due to its ability to target AT-rich regions, the generation of sticky ends after cleavage, and the simplified requirement of requiring only a single crRNA. However, existing Cas12a and its mutants have certain limitations in editing efficiency, PAM (protospacer adjacent motif) compatibility, and specificity, affecting their effectiveness and flexibility in broader genome editing applications, and urgently need improvement. Summary of the Invention

[0003] One objective of this invention is to provide the Cas12a homolog PsCas12a protein and its encoding gene;

[0004] The second objective of this invention is to provide a mutant of the Cas12a homolog PsCas12a protein and its encoding gene;

[0005] A third objective of this invention is to provide a vector containing the encoded gene and a host cell;

[0006] A fourth objective of this invention is to provide a CRISPR-Cas system comprising the Cas12a homolog PsCas12a protein or a mutant thereof;

[0007] The fourth objective of this invention is to apply the Cas12a homolog PsCas12a protein or its mutants, a vector containing the encoding gene, or a CRISPR-Cas system including the Cas12a homolog PsCas12a protein or its mutants to gene editing, editing target nucleic acids, gene cutting, target nucleic acid detection and / or diagnosis, or preparation of targeted gene therapy drugs.

[0008] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0009] One aspect of the present invention is to provide a Cas12a homolog, PsCas12a protein, the amino acid sequence of which is shown in SEQ ID No. 1.

[0010] This invention screens out the Cas12a homolog PsCas12a protein with potential gene editing capabilities through sequence screening and phylogenetic analysis, and demonstrates that PsCas12a protein has better editing effects than Cas12a protein in terms of gene editing efficiency, PAM compatibility or specificity.

[0011] A second aspect of the invention is to provide a mutant of the Cas12a homolog PsCas12a protein, said mutant being a single-point mutant obtained by performing a single-point mutation of any one of the following amino acids in the amino acid sequence shown in SEQ ID No. 1: V16L, M42R, M65L, S107I, D540N, S543T, F547Y, L588I, A742S, Q774E, N877D, I966V, N969K, A973M, H980Y, S993G, K995L, A1160Q, or Y1163N; preferably, SEQ ID No. The monoparticle mutant obtained by performing a single point mutation of any one of the amino acids V16L, M42R, M65L, S107I, D540N, S543T, L588I, Q774E, N877D, I966V, H980Y, or S993G on the amino acid sequence shown in SEQ ID No. 1; most preferably, the monoparticle mutant obtained by performing a single point mutation of any one of the amino acids V16L, D540N, or H980Y on the amino acid sequence shown in SEQ ID No. 1; wherein, the amino acid sequence of the monoparticle mutant obtained by performing a single point mutation of the amino acid sequence shown in SEQ ID No. 1 with the amino acid V16L amino acid is shown in SEQ ID No. 2, the amino acid sequence of the monoparticle mutant obtained by performing a single point mutation of the amino acid sequence shown in SEQ ID No. 1 with the amino acid D540N amino acid is shown in SEQ ID No. 3, and the amino acid sequence of the monoparticle mutant obtained by performing a single point mutation of the amino acid sequence shown in SEQ ID No. 1 with the amino acid H980Y amino acid is shown in SEQ ID No. 4.

[0012] In this invention, the single point mutant “V16L” represents the mutation of the 16th amino acid of the Cas12a homolog PsCas12a, whose amino acid sequence is shown in SEQ ID NO.1, from valine (Val, V) to leucine (Leu, L); the descriptions of the other single point mutants in this invention follow the same pattern.

[0013] Another aspect of the present invention provides a coding gene for the Cas12a homolog PsCas12a or a coding gene for a mutant of the Cas12a homolog PsCas12a.

[0014] Another aspect of the present invention is to provide a vector comprising the coding gene and a regulatory element operatively linked to the coding gene; wherein the vector may be an expression vector, a cloning vector, or a shuttle vector, etc.

[0015] In a preferred embodiment, the regulatory element is selected from one or more of promoters, terminators, enhancers, transposons, leader sequences, or marker genes.

[0016] Another aspect of the present invention is to provide a CRISPR-Cas system, the system comprising a Cas protein and at least one gRNA; the Cas protein is capable of binding the gRNA, the gRNA comprising a homologous repeat sequence and a spacer sequence capable of hybridizing with a target nucleic acid, wherein the Cas protein is the aforementioned Cas12a homolog PsCas12a protein or a mutant thereof.

[0017] Another aspect of the present invention provides a kit for gene editing or gene cutting, the kit comprising the above-mentioned Cas12a homolog PsCas12a protein or a mutant thereof, or a polynucleotide encoding the Cas12a homolog PsCas12a protein or a mutant thereof, or a vector containing the polynucleotide sequence thereof, or a CRISPR-Cas system containing the above-mentioned Cas12a homolog PsCas12a protein or a mutant thereof.

[0018] Another aspect of the present invention is to apply the Cas12a homolog PsCas12a protein or its mutant, or the polynucleotide encoding the Cas12a homolog PsCas12a protein or its mutant, or the vector containing the polynucleotide sequence, or the CRISPR-Cas system containing the above-mentioned Cas12a homolog PsCas12a protein or its mutant, or the kit for gene editing or gene cutting to gene editing, gene targeting, gene cutting, target nucleic acid detection and / or diagnosis or preparation of targeted gene therapy drugs.

[0019] In one specific embodiment of the present invention, the gene editing, gene targeting, or gene cutting is performed intracellularly and / or extracellularly; the gene editing or editing target nucleic acid includes modifying genes, knocking out genes, mutating genes, or changing the expression level of gene products, etc.

[0020] In one specific embodiment of the present invention, the gene editing, gene targeting, or gene cutting can be performed in prokaryotic or eukaryotic cells.

[0021] This invention discloses a novel Cas12a homolog, PsCas12a, derived from Paracoccus salsus. Compared to Cas12a, PsCas12a exhibits better editing performance in terms of gene editing efficiency, PAM compatibility, and specificity. Furthermore, through sequence screening and optimized design, this invention yields a series of PsCas12a mutants (including V16L, D540N, and H980Y) with even higher editing efficiency and expanded PAM compatibility. In vitro and in vivo cutting experiments demonstrate that these mutants exhibit better gene editing performance in genome editing. The PsCas12a and its mutants provided by this invention have broad application prospects in genome editing of plants, animals, and microorganisms.

[0022] Definitions of terms involved in this invention

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods, apparatus, and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, preferred methods, apparatus, and materials are now described.

[0024] The terms “polynucleotide” or “nucleotide” mean deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and their polymers, in single-stranded or double-stranded form. Unless specifically limited, the term covers nucleic acids containing known analogs of natural nucleotides, which have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, including PNAs (peptide nucleic acids), DNA analogs (phosphate thioesters, phosphoramidites, etc.) used in antisense techniques. Unless otherwise specified, a specific nucleic acid sequence also implicitly covers variants of its conserved modifications (including, but not limited to, degenerate codon substitutions) and complementary sequences, as well as explicitly specified sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the 3rd position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue (Mol Cell. Probes 8:91-98 (1994)).

[0025] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. That is, the description of a polypeptide is equally applicable to the description of a peptide and the description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the terms cover amino acid chains of any length, including full-length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.

[0026] The terms “mutation” and “mutant” have their common meanings here, referring to genetic, naturally occurring or introduced changes in a nucleic acid or polypeptide sequence, and their meanings are the same as those commonly known to those skilled in the art.

[0027] The terms "recombinant host cell line" or "host cell" refer to a cell containing the polynucleotides of the present invention, regardless of the method used for insertion to produce the recombinant host cell, such as direct uptake, transduction, f-pairing, or other methods known in the art. The exogenous polynucleotides may be maintained as, for example, non-integrating vectors of plasmids or may be integrated into the host genome. The host cell may be a prokaryotic or eukaryotic cell.

[0028] The term "operable connection" refers to a functional connection between two or more elements, which can be adjacent or non-adjacent.

[0029] "Target sequence" refers to a polynucleotide that is targeted by the guide sequence in gRNA, such as a sequence that is complementary to the guide sequence, where hybridization between the target sequence and the guide sequence will promote the formation of the CRISPR / Cas complex (including Cas protein and gRNA). Attached Figure Description

[0030] Figure 1. Screening and analysis results of PsCas12a sequences; A. E-Value distribution of 115 Cas12a sequences identified by Hmmerscan analysis; B. Phylogenetic tree of PsCas12a and other classic Cas12a proteins (FnCas12a, LbCas12a and AsCas12a), showing their kinship.

[0031] Figure 2. Results of in vitro cleavage activity and related characteristics analysis of PsCas12a; A. Verification of PsCas12a's cleavage activity against the BMLacZ DNA fragment; the nucleic acid bands at different substrate concentrations show complete cleavage of PsCas12a at 100 ng substrate (1200 bp and 500 bp); when the substrate amount is increased to 200 ng, bands of 1700 bp, 1200 bp, and 500 bp appear; B. Effect of the ratio of Cas12a to gRNA on cleavage efficiency; the cleavage bands of each ratio group have similar brightness, indicating that the excess of gRNA does not significantly improve cleavage efficiency; C. Prediction of PsCas12a's preference for different PAM sequences; D. Specificity analysis of cleavage sites of PsCas12a and AsCas12a for synthetic PAM libraries; PsCas12a is more specific for cleavage at specific sites, while AsCas12a shows cleavage activity at multiple sites.

[0032] Figure 3. Cleavage activity analysis of PsCas12a and its mutants; A. Distribution of conserved motifs and mutation sites on PsCas12a, 19 mutation sites identified by MUSCLE alignment and their conservation positions during evolution; B. qPCR detection principle for verifying the in vitro cleavage activity of PsCas12a and its mutants; C. Comparison of in vitro cleavage activity of PsCas12a and its mutants with commercial AsCas12a and LbCas12a; D. Schematic diagram of the in vivo cleavage activity verification principle of PsCas12a mutants in BMLacZ target cells; E. Growth of PsCas12a mutants in liquid culture medium. Based on the high-order structure of PsCas12a, it was divided into four parts (Domain1, Domain2, Domain3, Domain4), and the growth of mutants in different domains was shown. The red curve represents the negative control group, the orange curve represents wild-type PsCas12a, and the other colors represent PsCas12a mutants. The in vivo activity test results of F.PsCas12a mutants under gradient dilution conditions were also shown. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments described are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0034] Test materials and test methods

[0035] 1. Cas12a sequence collection and phylogenetic analysis

[0036] Literature related to Cas12a was retrieved from the PubMed database of the National Center for Biotechnology Information (NCBI), and 23 reported Cas12a homolog protein sequences with nuclease activity were collected (see Supplementary Material 1). Multiple sequence alignment of these 23 sequences was performed using the MUSCLE tool in the phylogenetic analysis software MEGA. A phylogenetic tree was then constructed using Neighbor-Joining (NJ), with all analyses using default parameters. Finally, the phylogenetic tree was refined using the TVBOT tool.

[0037] 2. Motif Function Annotation Based on Protein Structure

[0038] The manually reviewed FnCas12a protein sequence was obtained from the Uniprot database. Structure alignment was performed using the MatchMaker tool in Chimera software, and the correspondence between functional fragments and conserved motifs was annotated based on the protein structure.

[0039] 3. Sequence mining based on conserved motifs

[0040] HMMER software (DURBIN et al., 1998), a sequence analysis toolkit based on Hidden Markov Models (HMM), was used to search for homologous amino acids or nucleotide sequences. First, conserved motifs were extracted from 23 functional Cas12a sequences using the MEME online tool (BAILEY et al., 2015), with motif lengths set to 6-50 amino acids, and 30 motifs were extracted. Then, these conserved motif results were constructed into an HMM file, and the Hmmerscan tool was used to scan and search the original sequences, with an E-value set to e-4, to identify motifs commonly contained in these functional Cas12a sequences. Next, Hmmerscan was used to search for these conserved motifs in the UniRef100 database, with the search amino acid sequence length set between 500-2000 amino acids. The number of sequences containing different conserved motifs was counted, and the sequence containing the most motifs was selected.

[0041] 4. Sequence mining of novel Cas12a

[0042] Multiple sequence alignment was performed on 115 sequences using the MUSCLE tool in the phylogenetic analysis software MEGA. A phylogenetic tree was then constructed using the Maximum Likelihood (ML) method, with all analyses using default parameters. Finally, the phylogenetic tree was refined using the TVBOT tool.

[0043] 5. Protein structure comparison

[0044] The tertiary structure of PsCas12a was predicted using AlphaFold 3 (AF3). The protein structures of AsCas12a, LbCas12a, and FnCas12a were obtained from the PDB database and aligned using PyMOL.

[0045] 6. Construction of Cas12a expression plasmid

[0046] The plasmid pET-28a(-) was used for the soluble expression of the Cas12a protein. This plasmid was synthesized by General Biosciences, and the Cas12a gene sequence was optimized according to the codon preferences of *E. coli*. A stop codon (TAA) was added to the 3' end of the optimized gene sequence, and it was cloned into the pET-28a(-) vector at the insertion site between NdeI and XhoI. The synthesized plasmid was sequenced and verified for accuracy before delivery.

[0047] 7. Design and Synthesis of gRNA

[0048] To verify the in vitro cleavage activity of Cas12a, the mapple gene from the BMLacZ engineered strain was selected as the target. The genome sequence of *Paracoccus salsus* was downloaded from the NCBI database, and repeat sequences with high scores were screened using the CRISPRDetect tool (BISWAS et al., 2016). Since no suitable repeat sequence was found in this genome, the repeat sequence corresponding to MbCas12a, which is closely related to PsCas12a, was selected. A 20bp spacer (guide sequence) was designed targeting the PAM TTTG sequence on the mapple gene; this sequence is inversely complementary to the target site. Finally, a 40bp oligonucleotide gRNA was synthesized, and its sequence is as follows:

[0049] The gRNA, 5'-UAAUUUCUACUAUUGUAGAUUCUCCACAAUUUAUGUACGG-3', contains a 20bp spacer sequence and a 20bp crRNA-tracrRNA sequence. To prevent degradation of the gRNA in vitro, two thio and methoxy modifications (synthesized by GenScript) were added to the 5' and 3' ends. Additionally, the following oligonucleotides were synthesized for subsequent control experiments:

[0050] AsCas12a-gRNA:

[0051] 5'-UAAUUUCUACUCUUGUAGAUUCUCCACAAUUUAUGUACGG-3'

[0052] LbCas12a-gRNA:

[0053] 5'-UAAUUUCUACUAAGUGUAGAUUCUCCACAAUUUAUGUACGG-3'

[0054] 8. In vitro cleavage of double-stranded DNA

[0055] The in vitro cleavage reaction was performed in a 20 μL system. The reaction components included Cas12a (2 pmol), gRNA (6 pmol), 10×Cas12a reaction buffer (10 mM Tris-HCl, 300 mM NaCl, 0.5 mM DTT, 50% glycerol, pH 7.4, purchased from GenScript), DNA fragment (200 ng, adjusted as needed), and RNA-free H2O (to a final volume of 20 μL). The reaction mixture was added sequentially to the reaction tube, mixed thoroughly, and incubated at 37°C for 10 minutes to form the RNP (ribonucleoprotein) complex. Then, 200 ng of DNA substrate was added, gently mixed, and incubated at 37°C for 20 minutes to initiate the cleavage reaction. After the reaction was complete, 10 μL of terminator (0.5% SDS, 50 mmol / L EDTA, pH 8.0) was added to terminate the reaction. The reaction products were analyzed by agarose gel electrophoresis to evaluate the cleavage efficiency and specificity of the Cas12a-gRNA complex.

[0056] 9. Preparation of the cutting substrate

[0057] To obtain the cleavage substrate, upstream primer substrate-F and downstream primer substrate-R were designed and synthesized, and their concentrations were diluted to 10 μM. Using BMLacZ bacterial culture as a template, a 1700 bp double-stranded DNA fragment containing a mapple from the BMLacZ genome was amplified. The total volume of the amplification reaction system was 50 μL, including 25 μL of 2×Phanta Max Master Mix (Dye Plus) (Nanjing Novizan Biotechnology Co., Ltd.), 2 μL of upstream primer (10 μM), 2 μL of downstream primer (10 μM), 1 μL of template DNA, and 20 μL of RNA-free H2O. The amplification program was as follows: initial denaturation was performed at 95 °C for 3 minutes, followed by 35 cycles, each cycle consisting of denaturation at 95 °C for 15 seconds, annealing at 55 °C for 15 seconds, and extension at 72 °C for 100 seconds. The final extension was performed at 72 °C for 5 minutes, and the sample was stored at 4 °C. After the amplification reaction was completed, agarose gel electrophoresis was used to verify the nucleic acid fragments in the reaction solution. After confirming the target fragment by electrophoresis, it was purified and recovered to obtain the target fragment substrate for subsequent experiments.

[0058] 10. Construction of PAM-substituted DNA fragments

[0059] Site-directed mutagenesis was used to construct a substrate fragment containing atypical PAM. A forward and reverse primer for the mutated site was designed and synthesized at the target mutation site, with specific sequences shown in Table 1. These primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The initial substrate for the amplification system was the substrate described in section 2.8. Amplification was performed by binding the upstream primer substrate-F and the downstream primer substrate-R, generating two fragments. For example, primer TTTG-F was used to bind substrate-R, and primer TTTG-R was used to bind substrate-F to perform site-directed mutagenesis of the PAM sequence of TTTG. Then, gel extraction was performed to extract the two fragments, and appropriate amounts of these fragments were used as templates for amplification. The amplification system and procedure were the same as described in section 2.8. After obtaining the reaction solution containing the target fragment, an appropriate amount of the reaction solution was used as a template for further amplification using substrate-F and substrate-R. Finally, all the reaction solution was subjected to agarose gel electrophoresis, and the target substrate fragment with PAM replacement was obtained by gel extraction.

[0060] 11. Analysis of cleavage sites

[0061] Cleavage sites were detected using an in vitro DNA library assay. A 150 bp linear double-stranded DNA library containing four random deoxyribonucleotides and 256 different PAM sequences was synthesized and mixed in equal proportions. The target sequence was consistent with the sequence used for in vitro and in vivo assays (provided by Universal Biotech). 400 ng of DNA substrate was cleaved with 10 pmol Cas12a protein in a 20 μL reaction system at 37°C for 20 min. The reaction was terminated by adding 10 μM terminator (0.5% SDS and 50 mM EDTA, pH 8.0). The reaction system was then subjected to next-generation sequencing (performed by MegaBio), and bioinformatics methods were used to analyze PAM preference and cleavage sites associated with nuclease activity.

[0062] 12. In vitro genome dissection and qPCR analysis

[0063] Genomic DNA was extracted from BMLacZ and used as a substrate for in vitro cleavage. The cleavage reaction system consisted of 20 μL of Cas12a (2 pmol), gRNA (6 pmol), 10×Cas12a reaction buffer (10 mM Tris-HCl, 300 mM NaCl, 0.5 mM DTT, 50% glycerol, pH 7.4), genomic DNA (2000 ng), and RNA-free H2O to a final volume of 20 μL. First, Cas12a, gRNA, 10×Cas12a reaction buffer, and RNA-free H2O were incubated at 37°C for 10 minutes to form an RNP complex. Then, 2000 ng of genomic DNA was added, and the reaction was incubated at 37°C for 20 minutes to initiate the cleavage reaction. After the reaction, the enzyme was inactivated by heating at 95°C to terminate the reaction. The reaction product was diluted to 1 ng / μL and used as a template for real-time quantitative PCR (qPCR). The qPCR amplification system consisted of the following components: a total volume of 20 μL for each reaction, including 1 μL of diluted template (1 ng / μL), 0.4 μL of upstream primer (10 μM), 0.4 μL of downstream primer (10 μM), 10 μL of 2×Taq Pro Universal SYBR qPCR Master Mix (Nanjing Novizan Biotechnology Co., Ltd.), and 8.2 μL of RNA-free H2O. The qPCR reaction program was as follows: initial denaturation at 95℃ for 30 seconds, followed by 40 cycles, each cycle consisting of 10 seconds of denaturation at 95℃ and 30 seconds of annealing at 60℃.

[0064] 13. Construction of single-point mutants

[0065] Single-point mutants were constructed using a two-round PCR method. Specific primers were designed at the mutation site and amplified using universal primers T7 or T7-Ter for the pET-28a vector. The first round of PCR used the wild-type plasmid as a template to amplify a small fragment containing the mutation site. The second round of PCR used the first round PCR product as primers, again using the wild-type plasmid as a template, to amplify the entire plasmid. After amplification, residual wild-type plasmid template was removed by DpnI digestion. The digestion reaction was carried out at 37°C for 3 hours. The digestion system consisted of 50 μL of the second round PCR product, 5 μL of 10×cutsmart buffer, and 1 μL of DpnI enzyme.

[0066] After purification, the enzyme digestion products were transformed into Top10 competent cells (Nanjing Novizan Biotechnology Co., Ltd.) and cultured overnight at 37°C. Single colonies were picked and expanded. Using the bacterial culture as a template, PCR verification was performed using T7 and T7-Ter primers to confirm the presence of mutants. For positive colonies, first-generation sequencing was performed to verify the accuracy of the mutations (Beijing Qingke Biotechnology Co., Ltd.). Finally, the confirmed bacterial culture (preserved with an equal volume of 40% glycerol) and plasmids were stored.

[0067] 14. Construction of gRNA plasmid and gradient dilution culture of bacterial culture co-transformed with two plasmids

[0068] The gRNA plasmid was synthesized by General Biotechnology. Its sequence was identical to the in vitro synthesized gRNA, but with TTTTTT appended at the end as a transcription termination signal. The synthesized gRNA gene was cloned into the pACYCDuet-1 vector, inserted between the NcoI and XhoI restriction sites. After sequencing confirmation, the plasmid was delivered. 100 ng of different wild-type or mutant pET-28a-Cas12a plasmids were mixed with the pACYCDuet-1-gRNA plasmid and added to an equal volume (ensuring excess) of BMLacZ competent cells. After heat shock transformation at 42℃, the revived bacterial culture was serially diluted 2, 4, 6, 8, and 10-fold. The equal-volume diluted bacterial culture was vertically dropped onto square solid culture media containing 50 μg / mL kanamycin, 25 μg / mL chloramphenicol, and 0.3 mM IPTG. After drying, the culture was incubated upside down at 37℃ for 16 hours, and the results were observed.

[0069] 15. Determination of growth curves of strains after co-transformation with two plasmids in vivo

[0070] 50 ng each of pET-28a-Cas12a plasmid and pACYCDuet-1-gRNA plasmid were mixed and heat-shocked at 42°C to co-transform BMLacZ competent cells. The transformed bacterial culture was plated on LB agar containing kanamycin and chloramphenicol and incubated upside down at 37°C for 16 hours. Subsequently, a single transformed colony was picked from the medium and inoculated into a 96-well plate containing 200 μL of LB liquid medium (containing IPTG, kanamycin, and chloramphenicol) and continuously incubated at 37°C and 600 rpm on a horizontal shaker for 12 hours. The optical density (OD600) of the bacterial culture was measured at 600 nm every hour to monitor the growth curve of the strain.

[0071] Experimental Example 1: Discovery, evolutionary analysis, and sequence alignment analysis of PsCas12a, a homolog of Cas12a.

[0072] Literature related to Cas12a was retrieved from the NCBI database, and 23 Cas12a homolog sequences with nuclease activity were collected. Conserved motif analysis was then performed on these 23 sequences. A Hidden Markov Model (HMM) file was constructed based on these motifs, and further searches were conducted on the original sequences. The results identified 22 conserved motifs among these functional Cas12a sequences. Conserved motifs were extracted using HMMER software, and potential functional sequences were searched in the UniRef100 database, ultimately selecting 115 sequences containing all conserved motifs. The E-value of the 22 conserved motifs in these 115 sequences was further analyzed using the Hmmerscan tool. A lower E-value indicates a higher similarity between the target sequence and the conserved motif. The sequence with the lowest E-value (0.002), which was not previously reported in the literature, came from Cas12a of Paracoccus salsus (PsCas12a), with a sequence length of 1242 amino acid residues (Figure 1A). Multiple sequence alignment was performed on 115 sequences to construct a phylogenetic tree. Evolutionary analysis showed that PsCas12a is in an adjacent cluster with FnCas12a and LbCas12a, indicating a close phylogenetic relationship (Figure 1B). Sequence alignment of PsCas12a with the classic AsCas12a, LbCas12a, and FnCas12a showed similarity scores of 37.12%, 40.29%, and 42.85%, respectively.

[0073] The amino acid sequence of PsCas12a (Paracoccus salsus) is as follows:

[0074] Experimental Example 2: PsCas12a In Vitro Cleavage Activity Verification Test

[0075] To verify the nuclease activity of PsCas12a, a 1700bp DNA fragment from BMLacZ (an E. coli BL21(DE3) strain whose genome contains the red fluorescent protein gene mapple, enabling constitutive expression of red fluorescent protein) was selected as the in vitro cleavage substrate. The results, shown in Figure 2A, indicate that PsCas12a achieved complete cleavage with 100ng of substrate, producing two nucleic acid bands (1200bp and 500bp). Since the cleavage efficiency of the nuclease is constant over a certain period, the amount of uncleaved fragment (1700bp) gradually increased with increasing substrate quantity. When the substrate quantity exceeded 200ng, three nucleic acid bands of different sizes (1700bp, 1200bp, and 500bp) appeared. These results clearly demonstrate that PsCas12a possesses nuclease activity and can effectively cleave DNA fragments.

[0076] Experimental Example 3: Effect of the ratio of PsCas12a to gRNA on in vitro cleavage efficiency

[0077] This study investigated the effect of the Cas12a to gRNA ratio on cleavage efficiency. With the Cas12a amount kept constant at 2 pmol, three different Cas12a to gRNA ratios of 1:1, 1:2, and 1:3 were established, while other experimental conditions remained unchanged. The results are shown in Figure 2B. The band brightness was similar across groups, indicating that increasing gRNA did not significantly improve cleavage efficiency. In the PsCas12a in vitro cleavage experiment, an excess of gRNA achieved the optimal cleavage effect.

[0078] Experimental Example 4: PAM Preference Determination Test of PsCas12a

[0079] Substrate fragments containing atypical PAM sequences (TTTA, TTTC, TTTT, and TTTG) were constructed using site-directed mutagenesis and in vitro cleavage experiments were performed to analyze the PAM preference of PsCas12a.

[0080] The results of PAM preference determination of PsCas12a are shown in Figure 2C. PsCas12a can cleave target sequences with PAM of TTTV (A / C / G). The substrate with PAM of TTTG has the highest cleavage efficiency, which is reflected in the lowest band brightness. TTTC is the second highest, and TTTA has a low cleavage efficiency. The target sequence with PAM of TTTT is almost not cleaved.

[0081] In addition, this experiment also constructed substrate fragments with PAM sequences of TTCG, TCTG, CTTG, TCCG, CCTG, and CCCG. These sequences are usually difficult to target or have low cleavage efficiency by Cas12a. However, the detection results showed that PsCas12a also showed a certain preference for atypical PAM sequences (such as TTCG, TCTG, and CTTG), but the cleavage efficiency of PsCas12a was still the highest on substrates with PAM of TTTG.

[0082] Experimental Example 5: Detection of PsCas12a cleavage site specificity

[0083] The synthesized PAM library was cleaved using AsCas12a and PsCas12a, and the cleavage site specificity of PsCas12a was analyzed.

[0084] The cleavage site specificity test results of PsCas12a are shown in Figure 2D: PsCas12a showed significant cleavage between nucleotides 15 and 16 of the non-target strand, and also significant cleavage between nucleotides 20 and 21 of the target strand. In contrast, AsCas12a showed cleavage at multiple positions on the non-target strand, including positions 12 and 13, 14 and 15, 15 and 16, 16 and 17, and 17 and 18, and also cleavage between positions 20 and 21, and 21 and 22 of the target strand. These results indicate that PsCas12a has more specific cleavage sites, showing significant cleavage at only two positions, while AsCas12a shows significant cleavage at multiple positions. Therefore, PsCas12a is more suitable for achieving precise gene editing than AsCas12a.

[0085] Experimental Example 6: Design of PsCas12a mutant

[0086] This study used a homology sequence alignment method to identify and select conserved sites in PsCas12a for mutation design. By performing a muscle alignment of PsCas12a with the remaining 114 potential functional Cas12a sequences in the UniRef100 database, 19 amino acid sites were found to have high conservation during evolution. To ensure that the functionality of PsCas12a is consistent with these evolutionary trends, amino acids at corresponding positions were replaced with more frequently occurring residues, resulting in 19 single-point mutants: V16L, M42R, M65L, S107I, D540N, S543T, F547Y, L588I, A742S, Q774E, N877D, I966V, N969K, A973M, H980Y, S993G, K995L, A1160Q, and Y1163N. Except for S107I, A1160L, and Y1163N, the remaining mutation sites are all located within conserved motifs (Figure 3A).

[0087] Example 7: In vitro cleavage activity verification test of PsCas12a mutant

[0088] The relative cleavage efficiencies of various Cas nucleases were quantitatively compared by qPCR (real-time quantitative PCR) using the BMLacZ genome as an in vitro cleavage substrate.

[0089] The principle of qPCR detection is shown in Figure 3B. The experimental group used diluted cleavage products as templates for qPCR detection, while the control group used an equal amount of uncut genomic DNA as templates. In each reaction, two pairs of primers were used for amplification: primer N amplified fragments without cleavage sites, and primer Y amplified fragments containing cleavage sites. If the nuclease is active, the genomic DNA will break at the cleavage site, reducing the amount of template in primer Y and thus reducing the number of amplified fragments. The higher the activity of the nuclease, the larger the Ct value of primer Y, while the Ct value of primer N should be the same as the control group. By calculating the ΔΔCt value, the relative activities of each nuclease can be compared.

[0090] In addition, the activities of commercially available AsCas12a and LbCas12a were also tested in this experiment; the results are shown in Figure 3C, verifying that PsCas12a has significant activity. Among the 19 mutants designed and constructed, V16L, M42R, M65L, S107I, D540N, S543T, L588I, Q774E, N877D, I966V, H980Y, and S993G showed superior cleavage activity compared to wild-type PsCas12a, with V16L, D540N, and H980Y mutants exhibiting the best in vitro cleavage activity.

[0091] The amino acid sequence of mutant V16L is shown in SEQ ID No. 2:

[0092] The amino acid sequence of mutant D540N is shown in SEQ ID No. 2:

[0093] The amino acid sequence of mutant H980Y is shown in SEQ ID No. 4:

[0094] Experimental Example 8: In vivo cleavage activity verification experiment of PsCas12a mutant

[0095] This experiment utilized the fact that most prokaryotes lack a non-homologous end joining (NHEJ) repair system to construct a lethal system targeting the mapple gene on BMLacZ (Figure 3D). The results are shown in Figure 3E. The red curve represents the negative control group, which grew normally. The orange curve represents the wild-type PsCas12a bacterial culture density, which was lower than the negative control group, indicating that wild-type PsCas12a possesses nuclease activity. The mutant strains V16L, D540N, S543T, and H980Y had lower bacterial cultures density than wild-type PsCas12a, suggesting that these mutants may have higher editing efficiency in vivo. The growth of the remaining mutant strains was similar to or higher than that of wild-type PsCas12a, indicating that while these mutants possess nuclease activity, the activity was not significantly enhanced.

[0096] To further verify the in vivo activity of these mutants, wild-type and mutant plasmids were co-transformed with plasmids containing gRNA. The serially diluted co-transformed bacterial cultures were then spotted onto solid medium containing IPTG for incubation. The results are shown in Figure 3F. For all strains (containing different Cas12a plasmids), the colony count decreased with increasing dilution. Specifically, mutants V16L, D540N, S543T, A742S, I966V, N969K, and H980Y had fewer colonies at each dilution than wild-type PsCas12a, indicating that these mutants have superior in vivo cleavage activity compared to the wild type. In vitro and in vivo cleavage activity verification of the PsCas12a mutants showed that mutants V16L, D540N, and H980Y exhibited excellent editing activity in both in vitro and in vivo cleavage activity tests.

Claims

PsCas12a, a homolog of Cas12a, is characterized by: Its amino acid sequence is shown in SEQ ID No.

1. The mutant of the homolog PsCas12a according to claim 1 is characterized in that, The mutant is obtained by performing a single-point mutation on any one of the amino acid sequences shown in SEQ ID No. 1, namely V16L, M42R, M65L, S107I, D540N, S543T, F547Y, L588I, A742S, Q774E, N877D, I966V, N969K, A973M, H980Y, S993G, K995L, A1160Q, or Y1163N; preferably, a single-point mutant obtained by performing a single-point mutation on any one of the amino acid sequences shown in SEQ ID No. 1, namely V16L, M42R, M65L, S107I, D540N, S543T, L588I, Q774E, N877D, I966V, H980Y, or S993G; most preferably, a single-point mutant obtained by performing a single-point mutation on any one of the amino acid sequences shown in SEQ ID No. 1, namely V16L, M42R, M65L, S107I, D540N, S543T, L588I, Q774E, N877D, I966V, H980Y, or S993G; most preferably, a single-point mutant obtained by performing a single-point mutation on any one of the amino acid sequences shown in SEQ ID No. 1, namely V16L, M42R, M65L, S107I, D540N, S543T, L588I, Q774E, N877D, I966V, H980Y, or S993G; The amino acid sequence shown in SEQ ID No. 1 is a single point mutant obtained by performing a single point mutation of any one of the amino acids V16L, D540N, or H980Y; wherein, the amino acid sequence of the single point mutant obtained by performing a single point mutation of the amino acid sequence shown in SEQ ID No. 1 on the V16L amino acid is shown in SEQ ID No. 2, the amino acid sequence of the single point mutant obtained by performing a single point mutation of the amino acid sequence shown in SEQ ID No. 1 on the D540N amino acid is shown in SEQ ID No. 3, and the amino acid sequence of the single point mutant obtained by performing a single point mutation of the amino acid sequence shown in SEQ ID No. 1 on the H980Y amino acid is shown in SEQ ID No.

4. The gene encoding the homolog PsCas12a of claim 1. The gene encoding the homolog PsCas12a of claim 2. A carrier, characterized in that, The vector comprises the coding gene as described in claim 3 or 4 and a regulatory element operatively linked to the coding gene. The carrier according to claim 5, characterized in that, The vector is selected from expression vectors, cloning vectors, or shuttle vectors. A CRISPR-Cas system comprising a Cas protein and at least one gRNA; the Cas protein is capable of binding the gRNA, the gRNA comprising a homologous repeat sequence and a spacer sequence capable of hybridizing with a target nucleic acid, characterized in that... The Cas protein is the homolog PsCas12a of claim 1 or the mutant of claim 2. A kit for gene editing or gene cutting, characterized in that, The kit includes the homolog PsCas12a of claim 1, the mutant of claim 2, the coding gene of claim 3 or 4, the vector of claim 5 or 6, or the CRISPR-Cas system of claim 7. The application of the homolog PsCas12a of claim 1, the mutant of claim 2, the encoding gene of claim 3 or 4, the vector of claim 5 or 6, or the CRISPR-Cas system of claim 7 in gene editing, editing target nucleic acid, gene cutting, target nucleic acid detection and / or diagnosis, or in the preparation of targeted gene therapy drugs. The application according to claim 9 is characterized in that, The gene editing or editing target nucleic acid includes modifying genes, knocking out genes, mutating genes, or changing the expression level of gene products; the gene editing, editing target nucleic acid, or gene cutting is performed in prokaryotic or eukaryotic cells.

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