A yeast ternary CRISPR-Cas9 system for screening Cas9 protein mutants and a screening method thereof

Abstract

The application belongs to the technical field of bioengineering, and particularly relates to a yeast ternary CRISPR-Cas9 system for screening Cas9 protein mutants and a screening method thereof.The yeast ternary CRISPR-Cas9 system is composed of an sgRNA expression vector, a Cas protein mutant screening vector and a reporter vector. The yeast ternary CRISPR-Cas9 system and the screening method provided by the application can obtain Cas9 mutants that can efficiently screen and cut various target sites, and expand the application field and editing efficiency of the CRISPR gene editing system.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a yeast ternary CRISPR-Cas9 system and its screening method for screening Cas9 protein mutants. Background Technology

[0002] In 1987, scientists discovered E. coli iap The discovery of a homologous sequence at the 3' end of a gene marks the first systematic identification of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and its associated protein (Cas). The CRISPR system, an adaptive immune defense mechanism in bacteria and archaea, protects these microorganisms from foreign nucleic acids. CRISPR consists of a series of short DNA repeats and unique spacer sequences inserted within them, derived from genetic material from previous viral infections or plasmid invasions. Through this mechanism, bacteria and archaea can recognize and defend against similar invasive nucleic acids using the CRISPR system. This discovery spurred scientists to delve deeper into the workings of the CRISPR-Cas system, ultimately laying the foundation for the rapid development of genome editing technology. In 2012, scientists revealed the gene-editing potential of the Cas9 protein in the CRISPR / Cas9 system, marking a breakthrough in the field of biotechnology. The CRISPR / Cas9 system, through the synergistic action of guide RNA (gRNA) and the Cas9 nuclease, can create double-strand breaks in specific DNA sequences, thereby activating the cell's repair mechanisms to perform gene knockout, insertion, or replacement operations. This technology has a wide range of applications, covering multiple fields such as basic scientific research, agricultural breeding, and disease treatment.

[0003] CRISPR-Cas systems exist in various types in bacteria and archaea, among which the Class 2 CRISPR-Cas system has been widely used in genome editing due to its high efficiency and simplicity. In particular, the Type II system utilizes Streptococcus pyogenes (Streptococcus pyogenes). Streptococcus pyogenes The Cas9 protein in CRISPR, used as a gene-editing tool, possesses high specificity, low side effects, and high editing efficiency, making it the most commonly used CRISPR system. The associated Cas9 nuclease binds to gRNA to form a ribonucleoprotein (RNP) complex, guiding Cas9 to locate and cleave target DNA sequences. Through precise genome editing, CRISPR / Cas9 technology can achieve targeted modification of genetic information in cells or organisms, thereby achieving purposes such as treating genetic diseases and improving crops.

[0004] Although CRISPR / Cas9 technology has shown great potential in gene editing, it also has some limitations in its application: (1) Lack of Cas9 mutant screening platforms: There is a lack of systematic Cas9 random mutant library screening platforms. (2) Limited target sites: The strict requirements for NGG sequences limit the identification of suitable genomic targets, affecting the editing of key genes and the application of gene function research and disease treatment. (3) Difficulty in editing regulatory elements: Regulatory elements such as enhancers and silencers often have conserved sequences, but NGG sites may not be able to dock with them properly, increasing the difficulty of precise editing and thus affecting the study of gene expression regulation. (4) Changes in binding and cleavage efficiency: The relative position of NGG sites and target sequences affects the binding and cleavage efficiency of Cas9. Positions that are too far apart or too close together will reduce editing efficiency, especially when targeting long non-coding RNAs, because it is difficult to find suitable PAM sites. (5) Complexity of multiple editing: Identifying multiple NGG sites to be close to each target gene increases the difficulty of designing single guide RNA (sgRNA). As the number of genes to be edited increases, finding compatible PAM sites becomes more difficult, limiting its application in the treatment of polygenic diseases and complex biological research. Therefore, it is essential to develop a Cas9 random mutation library screening system for efficient screening of Cas9 mutants capable of cleaving various target sites. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a yeast ternary CRISPR-Cas9 system and screening method for screening Cas9 protein mutants. Using the yeast ternary CRISPR-Cas9 system and screening method provided by this invention, Cas9 mutants that can efficiently screen for cleavage of various target sites can be obtained.

[0006] To achieve the above objectives, the specific technical solution of the present invention is as follows: The first aspect of this invention provides a yeast ternary CRISPR-Cas9 system for screening Cas9 protein mutants. The yeast ternary CRISPR-Cas9 system consists of an sgRNA expression vector, a Cas protein mutant screening vector, and a reporter vector. The sgRNA expression vector is an expression vector containing the yeast strong promoter pADH1 to drive sgRNA transcription. The reporter vector is a vector containing a reporter gene. The Cas protein mutant screening vector is pGADT7-Cas9.

[0007] The pGADT7-Cas9 uses the pGADT7-Rec2 vector as its backbone vector, by replacing the GAL DNA-AD domain fragment in the pGADT7-Rec2 vector with... SpCas9The gene, i.e., is obtained; the GALDNA-AD domain is the fragment from the 5' to the 3' end of the pGADT7-Rec2 vector, specifically from 1554 bp to 2037 bp; the... SpCas9 The gene's NCBI accession number is 69900935.

[0008] Furthermore, the sgRNA expression vector is constructed using the expression vector pHIS2 as a backbone by replacing the gene fragments from the HIS3 promoter to the HIS3 terminator in the expression vector pHIS2 with the pADH1-gRNA expression cassette; the gene fragments from the HIS3 promoter to the HIS3 terminator are the gene fragments from the 9th bp to the 1450th bp from the 5' to the 3' end in the expression vector pHIS2.

[0009] Furthermore, the report carrier uses the pGBKT7 skeleton carrier.

[0010] Furthermore, the reporter gene is a transcription factor with self-activating activity, an resistance selection marker gene, or a auxotroph selection marker gene.

[0011] Furthermore, the reporter gene is BpEBP1 Genes; the stated BpEBP1 The gene's GenBank number is PP081490.

[0012] Furthermore, the yeast is the Y2HGold yeast strain.

[0013] A second aspect of the present invention provides a method for constructing a Cas9 protein mutant library using the system described above. Using the Cas9 protein mutant screening vector pGADT7-Cas9 described above as a template, random mutations targeting the Cas9 protein are introduced into the template by designing mutation primers to construct a Cas9 protein mutant library.

[0014] Furthermore, it includes the following steps: The DNA segment in the Cas9 protein-encoded nucleotide sequence of the pGADT7-Cas9 vector was deleted to obtain a linearized vector fragment; the DNA segment was the 4835bp~4925bp fragment in the Cas9 protein-encoded nucleotide sequence.

[0015] A DNA fragment containing a random nucleotide sequence is synthesized; the DNA fragment containing the random nucleotide sequence consists of the random nucleotide sequence and homologous arms at both ends of the random nucleotide sequence; the homologous arms are complementary to the adjacent sequences on both sides of the DNA segment deleted in the pGADT7-Cas9 vector. The linearized vector fragment was co-transformed with a DNA fragment containing random nucleotide sequences into yeast competent cells; By homologous recombination, the random nucleotide sequence is integrated into the location of the deleted DNA segment in the pGADT7-Cas9 vector to obtain a transformant population containing diverse Cas9 protein-coding sequences; The transformed subpopulation was cultured and collected to construct a Cas9 protein mutant library.

[0016] Furthermore, the homologous arm has a sequence length of 15 base pairs; the random nucleotide has a sequence length of 90 base pairs, used to introduce diverse mutations into the PAM interaction domain of the Cas9 protein.

[0017] A third aspect of this invention provides a method for screening non-NGG-dependent Cas9 protein mutants using the yeast ternary CRISPR-Cas9 system described above, comprising the following steps: Using the Cas protein mutant screening vector pGADT7-Cas9 as a template, random mutations targeting the Cas9 protein were introduced into the template by designing mutation primers, and a Cas9 protein mutant library was constructed. The plasmid of the Cas9 protein mutant library was transformed into yeast cells to obtain yeast cells containing the Cas9 mutant library; The reporter vector and sgRNA expression vector were co-transformed into the yeast cells, and the resulting positive monoclonal colonies were the non-NGG-dependent Cas9 mutants. Transformed yeast cells were cultured, and monoclonal colonies that could activate the reporter gene were screened. After verification, non-NGG-dependent Cas9 mutants were obtained.

[0018] Furthermore, the report carrier is pGBKT7- BpEBP1 The pGBKT7- BpEBP1 It is by BpEBP1 Obtained by cloning into the pGBKT7 vector; BpEBP1 The GenBank number is PP081490.

[0019] Furthermore, the non-NGG-dependent Cas9 mutant was obtained by culturing and screening in SD / -Trp / -URA / -LEU / +x-α-gal medium.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a yeast ternary CRISPR-Cas9 system and screening method for screening Cas9 protein mutants. This yeast ternary CRISPR-Cas9 system and screening method can efficiently screen for protein mutants that meet various requirements. 1) Optimization for inefficient targets: The yeast ternary CRISPR-Cas9 system of the present invention can screen Cas9 mutants that exhibit high cleavage ability on inefficient targets (such as target sequences with low cleavage efficiency), which significantly improves gene editing efficiency, especially when targeting genes or genomic regions that are difficult to cut.

[0021] 2) Identification of non-NGG PAM sites: Traditional CRISPR-Cas9 systems typically rely on NGG PAM sites, which limits the system's application scope. The screening method of this invention can rapidly identify and screen Cas9 mutants that function effectively at non-NGG PAM sites, enabling precise editing of genes lacking NGG PAM sites and expanding the target range of gene editing.

[0022] 3) High-efficiency screening in yeast: This system uses yeast strains as screening vectors to directly detect the cleavage efficiency of Cas9 protein at target sites in yeast, avoiding the inconvenience and complex operation of traditional screening methods using cell lines or other organisms, thus achieving high-throughput and rapid screening.

[0023] 4) Improve the flexibility of multi-gene editing: With the diversification of gene editing needs, especially for multi-gene editing tasks, this invention can obtain Cas9 mutants suitable for multiple gene targets through screening, which significantly simplifies the multiple editing design and provides more gene-targeted editing options.

[0024] 5) Improved system compatibility and universality: The yeast ternary CRISPR-Cas9 system provided by this invention can not only be widely used for screening different types of Cas9 mutants, but can also be extended to other CRISPR / Cas systems. It has strong universality and operability and is suitable for gene editing applications in a variety of species.

[0025] 6) Promoting the further development of CRISPR technology: The technology of this invention provides a new approach to genome editing technology, especially in the research of non-NGG-dependent CRISPR technology, providing a feasible screening platform, promoting the progress of CRISPR technology, and opening up new possibilities for applications in fields such as polygenic disease treatment, agricultural improvement and precision medicine.

[0026] In summary, the yeast ternary CRISPR-Cas9 system and screening method provided by this invention effectively solve some of the limitations of existing technologies and have broad application prospects, especially in multi-target editing, non-NGG site cleavage and efficient screening of mutants, providing important guarantees for the accuracy, efficiency and diversity of gene editing technology. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This describes the technical route for constructing the pGADT7-Cas9-M mutant library. Figure 1 'a' in the context of 'use' Sma Linearization of the pGADT7 plasmid was performed using restriction endonuclease I, followed by homologous recombination with yeast. SpCas9 The pGADT7-Cas9 vector was constructed by inserting it into the vector. Figure 1 b: Synthesize random primers to construct a 90bp random DNA library for mutating specific segments of the pGADT7-Cas9-M9 plasmid. In this example, the 4835bp-4924bp segment is used as an example for mutation. Figure 1 c: The pGADT7-Cas9-M9 plasmid was linearly amplified by reverse PCR to obtain a vector fragment of 4939bp-4820bp. Figure 1 In the d section: a double-stranded random DNA library and a linearized vector are co-transformed into yeast cells, and a Cas9 mutant library is established through yeast homologous recombination.

[0029] Figure 2 This is a schematic diagram illustrating the principle of Cas9 mutant screening that does not rely on NGG PAM sites. Figure 2 'a' in the text refers to the construction principle of the mutant library. Figure 2 b in the text refers to the construction of the screening system. Figure 2 c in the text refers to the screening of Cas9 mutants.

[0030] Figure 3 The cleavage of BpEBP1 by Cas9-M9 leads to mutations in the target gene and loss of its self-activation ability. Figure 3 In section a: Seven white colonies obtained from library screening were validated by spot assays on SD / -Leu / -Trp / -Ura / +X-α-gal and SD / -Leu / -Trp / -Ura / -His / -Ade+X-α-gal media. The bacterial suspensions were prepared at a ratio of 10... -1 10 -2 10 -3 Serial dilution. "C" is the negative control, containing only pGBKT7- BpEBP1Two plasmids, pHIS2-Target2-2-gRNA and M1-4, M6, M9, and M10, were selected from seven white clones. Figure 3 b: Sanger sequencing was performed on the Target2-2 site in the plasmid of the M9 mutant strain BD-BpEBP1 to determine whether the site had been edited. Figure 3 c in the figure represents the protein translation sequence of BpEBP1 after gene editing. Figure 3 In section d: Verification of the transcriptional activation activity of the mutated BpEBP1 sequence in the M9 clone. Due to the mutation, this protein no longer possesses transcriptional activation capabilities.

[0031] Figure 4 Structural analysis of the Cas9 mutant protein in the M9 mutant clone. Figure 4 In the image 'a': Details of the mutations in the Cas9 clone in M9. Site 1107E is a key amino acid for Cas9 to recognize the PAM region. In M9, Cas9-M9 experiences premature termination at amino acid 1099, resulting in premature termination of the Cas9 protein sequence. Figure 4 b: Schematic diagram of the backbone structure of the Cas9-M9-sgRNA-DNA complex. Figure 4 c: Schematic diagram of the surface structure of the Cas9-M9-sgRNA-DNA complex. Figure 4 The specific interaction between mutant amino acids and bases in d:Cas9-M9.

[0032] Figure 5 Comprehensive functional analysis of DNA cleavage efficiency of CRISPR-Cas9 M9 variants in all NNN PAM configurations. Figure 5 In the text 'a': the arrangement of different PAM site clones on the culture medium. Figure 5 b: Growth of clones with different PAM sites on SD / -Leu / -Trp / -Ura medium. Figure 5 c: Growth of clones with different PAM sites on SD / -Leu / -Trp / -Ura+X-α-gal medium. Figure 5 d: Growth of clones with different PAM sites on SD / -Leu / -Trp / -Ura / -His+X-α-gal medium. Detailed Implementation

[0033] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0034] Example 1: A yeast ternary CRISPR-Cas9 system and screening method for screening Cas9 protein mutants I. Experimental Methods 1. Construction of a yeast ternary CRISPR-Cas9 system To establish a Cas9 mutant screening system based on a yeast platform, this invention uses three backbone vectors: pHIS2, pGADT7-Rec2, and pGBKT7, with Y2HGold yeast strain as the host.

[0035] The yeast ternary CRISPR-Cas9 system consists of an sgRNA expression vector, a Cas protein mutant screening vector, and a reporter vector.

[0036] Taking the pHIS2-gRNA expression vector as an example, the construction method of the sgRNA expression vector is as follows: In the expression vector pHIS2, using Sma I and BamH The HIS3 promoter-HIS3 terminator fragment (9 bp~1450 bp) was excised by restriction endonuclease to obtain a linearized vector, which was then ligated to the pADH1-gRNA expression cassette to construct the pHIS2-gRNA vector.

[0037] Construction of the Cas protein mutant selection vector pGADT7-Cas9: via Kpn I and Sma The pGADT7-Rec2 vector was digested with enzyme I, its GALDNA-AD domain (1554 bp ~ 2037 bp) was deleted, and the inserted vector was replaced with... SpCas9 The gene sequence (NCBI gene accession number 69900935) was used to construct the pGADT7-Cas9 vector.

[0038] The report is carried in pGBKT7- BpEBP1 Taking a vector as an example, the construction method is as follows: The birch transcription factor with self-activation ability is used... BpEBP1 (GenBank accession number PP081490) was cloned downstream of GAL4 DNA-BD into the pGBKT7 vector to construct the pGBKT7- BpEBP1 Carrier.

[0039] 2. Construction of Cas9 mutant libraries based on yeast ternary systems The pGADT7-Cas9 plasmid was amplified by reverse PCR, and the 4835 bp~4925 bp region (corresponding to the PAM-interacting domain, the N-terminus of PID and its adjacent region) was removed from Cas9 to obtain the linearized vector pGADT7-Cas9-rPCR (primer sequences are shown in Table 1). Subsequently, a 90-base random DNA sequence (90N) was designed, with 15 bp sequences at both ends complementary to the DNA regions near amino acids 1092N and 1022R of Cas9 (primer information is shown in Table 1). This random sequence and the linearized vector pGADT7-Cas9-rPCR were co-transformed into Y2HGold yeast competent cells, and plated on 40 15cm plates containing SD / -Leu. The resulting colonies were collected to construct a Cas9 mutant library. The principles and steps of vector and library construction are described in [link to documentation]. Figure 1 .

[0040] Table 1 Primer information for constructing Cas9 mutant libraries in yeast ternary systems Subsequently, based on BpEBP1 The target sequence Target2-2, containing non-NGG PAM, was selected (primer information is shown in Table 2), and Golden Gate molecular cloning technology was used to introduce it upstream of the gRNA. Bsa I restriction site. Target2-2 was cloned into the enzyme... Bsa The linearized pHIS2-gRNA vector (pHIS2-Target2-2-gRNA) was then used to co-transform pHIS2-Target2-2-gRNA and pGBKT7-BpEBP1 into yeast cells containing the mutant library. The cells were plated on SD / -Leu / -Trp / -URA+x-α-gal plates. White colonies (potentially possessing Cas9 cleavage activity) were screened, and their pGBKT7 plasmids were extracted and Sanger sequenced to confirm the BpEBP1 mutation. Subsequently, the pGADT7 vector was extracted, and its Cas9 mutation region was sequenced to obtain the mutant sequence (named Cas9-M9).

[0041] Table 2. Non-NGG T2-2 target primers used for screening Cas9 mutants. 3. Yeast spot test and colony counting Yeast clones used for validation were cultured in SD / -Trp / -Leu / -URA liquid medium until the culture volume reached OD.600 =1.0. The bacterial suspension was then diluted 10... -1 times, 10 -2 times, 10 -3 Take 1.5 µL of the sample and spot it onto SD / -Trp / -Leu / -URA+x-α-gal and SD / -Trp / -Leu / -URA / -His / -Ade+x-α-gal solid medium, and incubate at 37°C for 3 days. Use ImageJ software to count the total number of colonies.

[0042] To further verify the cleavage ability of Cas9-M9 on target DNA at 64 different PAM sites, clones with Cas9 activity were selected from white colonies and cultured to OD200. 600 =0.8, diluted to 10 -1 Spot experiments were conducted on three types of culture media (SD / -Trp / -Leu / -URA solid medium, SD / -Trp / -Leu / -URA+x-α-gal solid medium, and SD / -Trp / -Leu / -URA / -His / -Ade+x-α-gal solid medium), and the media were incubated at 37°C for 3 days.

[0043] 4. Structural prediction of the Cas9-M9-target-gRNA complex The ternary complex structure of Cas9-M9-target DNA-gRNA was predicted using Alpha Fold3 and visualized using PyMOL (Version 3.0, Schrödinger, LLC). Domain color coding is as follows: RuvC domain: cyan; BH domain: green; REC1 domain: gray; REC2 domain: orange; HNH domain: bright pink; PI (PAMinteraction) domain: purple; target DNA: yellow; gRNA: dark blue. The model uses banded and surface representations, with mutated residues in Cas9-M9 highlighted in red.

[0044] II. Experimental Results 1. Construction and screening principle of a yeast ternary CRISPR screening system This invention constructs a yeast ternary CRISPR-Cas9 system to screen for Cas9 protein mutants capable of detaching from the classic "NGG" PAM. The yeast ternary CRISPR-Cas9 system includes a gRNA, a Cas9 protein, and a reporter gene (as expressed in the original text). BpEBP1(Taking as an example) First, the pHIS2 vector was modified. Since both pHIS2 and pGBKT7 use the TRP1 marker, the selection marker for pHIS2 was changed to URA3 to avoid marker conflict. Then, the gRNA sequence and the yeast strong promoter ADH1 (pADH1) were cloned into the modified pHIS2 to form the gRNA expression vector pHIS2-gRNA. Next, the pADH1-driven Cas9 gene was cloned into the pGADT7 vector to construct the Cas9 expression vector pGADT7-Cas9. To screen for Cas9 protein mutants, the birch BpEBP1 gene, which has self-activation capabilities, was cloned into pGBKT7 and transformed into Y2HGold, causing it to form blue colonies on a medium containing x-α-gal.

[0045] Since it was not possible to systematically mutate the entire PAM-interacting domain (PID), this study targeted the region (1093N-1122R) containing the key amino acid 1107E (which contacts dC-2 in N1G2G3 PAM), which is closely related to PAM binding. This region includes 1107E, part of RuvCIII, and the N-terminal sequence of PID. By replacing this 30aa segment entirely with a 30aa random sequence (corresponding to 90 random bases) and constructing it into the pGADT7 vector, a Cas9 random mutation library was formed. Figure 2 (a) in the middle.

[0046] Subsequently, the target BpEBP1 Furthermore, it has a non-NGG PAM-specific Target2-2 target site inserted into pHIS2-gRNA, followed by further insertion of pHIS2-T2-2-gRNA with pGBKT7- BpEBP1 Together they were transformed into yeast cells in the library ( Figure 2 (b) If the Cas9 mutant can cleave this non-NGG target site, the yeast colony will be white. The Cas9 mutant sequence can be obtained by extracting pGADT7 from the white colony and sequencing it. Figure 2 (c in the text)

[0047] 2. Obtain Cas9 mutants that do not depend on NGG PAM After the reporter vector and sgRNA expression vector were co-transformed into yeast cells containing the Cas9 mutant library, approximately 76,000 clones were obtained on 40 15cm plates, from which 7 white colonies were screened. Further validation on selective media showed that only the mutant M9 remained white on SD / -Trp / -URA / -Leu plates containing x-α-gal, and failed to grow at all on the more restrictive SD / -Trp / -URA / -Leu / -His2 / -Ade / +x-α-gal plates. Figure 3 (a) in the middle.

[0048] To confirm BpEBP1 Whether it was cut, pGBKT7 was extracted from the mutant M9 clone and sequenced, revealing a 1bp deletion within the Target2-2 region, resulting in a frameshift mutation. Figure 3 (b~c) The expression product loses its transcriptional activation ability ( Figure 3 (d in the text). Therefore, it is inferred that the Cas9 mutant in mutant M9 has the ability to cleave non-NGG PAM targets, and this mutant is named Cas9-M9.

[0049] 3. Cas9-M9 mutation sequence analysis Sequencing of pGADT7 extracted from mutant M9 revealed that the original sequence “NIVKKTE” in its Cas9 randomized region was replaced by “WSGQNG”, followed by a stop codon. Figure 4 The term "a" in the original text causes premature termination of translation in the Cas9 PI domain, resulting in Cas9-M9 lacking a complete PI domain. Structural prediction results show that Cas9-M9 completely lacks the PI domain. Figure 4 The mutation sites Trp-1093, Ser-1094, and Gly-1095 may form new hydrogen bonds with Asn-46, Ile-48, and Ala-50 in the RuvC1 region. Figure 4 (d) However, the REC and NUC regions of Cas9-M9 retain their intact structure, the HNH and RuvC catalytic centers function stably, and their gRNA-DNA binding ability remains unaffected. Therefore, despite the absence of the PI domain, Cas9-M9 can still achieve PAM-independent cleavage through structural stability and a novel hydrogen bond network.

[0050] 4. Cutting capability of Cas9-M9 on 64 types of PAM (NNN) To verify the PAM-independent nature of Cas9-M9, 64 NNN PAM-corresponding target sequences were constructed into pHIS2-gRNA and co-transformed into mutant M9 yeast cells using a reporter vector. All transformants grew normally on SD / -Trp / -URA / -Leu plates. Figure 5 (b) No blue color was observed on SD / -Trp / -URA / -Leu plates containing x-α-gal ( Figure 5 (c) and no growth was observed on the more stringent SD / -Trp / -URA / -Leu / -His2 / -Ade / +x-α-gal plates. Figure 5 (d) indicates that all target sites were cut by Cas9-M9.

[0051] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0052] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A yeast ternary CRISPR-Cas9 system for screening Cas9 protein mutants, characterized in that, The yeast ternary CRISPR-Cas9 system consists of an sgRNA expression vector, a Cas protein mutant selection vector, and a reporter vector; the sgRNA expression vector is an expression vector containing the yeast strong promoter pADH1; the reporter vector is a vector containing a reporter gene; and the Cas protein mutant selection vector is pGADT7-Cas9. The pGADT7-Cas9 uses the pGADT7-Rec2 vector as its backbone vector, and is modified by replacing the GALDNA-AD domain in the pGADT7-Rec2 vector with... SpCas9 The gene was obtained after gene sequencing; the GAL DNA-AD domain is the fragment from the 5' to the 3' end, specifically from 1554 bp to 2037 bp, in the pGADT7-Rec2 vector; SpCas9 The gene's NCBI accession number is 69900935.

2. The yeast ternary CRISPR-Cas9 system for screening Cas9 protein mutants according to claim 1, characterized in that, The sgRNA expression vector is constructed using the expression vector pHIS2 as a backbone by replacing the gene fragments from the HIS3 promoter to the HIS3 terminator in the expression vector pHIS2 with the pADH1-gRNA expression cassette; the gene fragments from the HIS3 promoter to the HIS3 terminator are the gene fragments from the 9th bp to the 1450th bp from the 5' to the 3' end in the expression vector pHIS2.

3. The yeast ternary CRISPR-Cas9 system for screening Cas9 protein mutants according to claim 1, characterized in that, The report carrier uses pGBKT7 as its backbone carrier.

4. The yeast ternary CRISPR-Cas9 system for screening Cas9 protein mutants according to claim 1, characterized in that, The reporter gene is a transcription factor with self-activating activity, an resistance selection marker gene, or a auxotroph selection marker gene.

5. The yeast ternary CRISPR-Cas9 system for screening Cas9 protein mutants according to claim 4, characterized in that, The reporter gene is BpEBP1 Genes; the stated BpEBP1 The gene's GenBank number is PP081490.

6. A method for constructing a Cas9 protein mutant library using the yeast ternary CRISPR-Cas9 system as described in claim 1, characterized in that, Using the Cas protein mutant screening vector pGADT7-Cas9 as a template, random mutations targeting the Cas9 protein were introduced into the template by designing mutation primers, and a Cas9 protein mutant library was constructed.

7. The construction method according to claim 6, characterized in that, Includes the following steps: The DNA segment in the Cas9 protein-encoded nucleotide sequence of the pGADT7-Cas9 vector was deleted to obtain a linearized vector fragment; the DNA segment was the 4835bp~4925bp fragment in the Cas9 protein-encoded nucleotide sequence.

8. Synthesize a DNA fragment containing a random nucleotide sequence; the DNA fragment containing the random nucleotide sequence consists of a random nucleotide sequence and homologous arms at both ends of the random nucleotide sequence; the homologous arms are complementary to the adjacent sequences on both sides of the DNA segment deleted in the pGADT7-Cas9 vector; The linearized vector fragment was co-transformed with a DNA fragment containing random nucleotide sequences into yeast competent cells; By homologous recombination, the random nucleotide sequence is integrated into the location of the deleted DNA segment in the pGADT7-Cas9 vector to obtain a transformant population containing diverse Cas9 protein-coding sequences; The transformed subpopulation was cultured and collected to construct a Cas9 protein mutant library.

9. The construction method according to claim 7, characterized in that, The homologous arm has a sequence length of 15 base pairs; the random nucleotide has a sequence length of 90 base pairs.

10. A method for screening non-NGG-dependent Cas9 protein mutants using the yeast ternary CRISPR-Cas9 system of claim 1, characterized in that, Includes the following steps: Using the Cas protein mutant screening vector pGADT7-Cas9 as a template, random mutations targeting the Cas9 protein were introduced into the template by designing mutation primers to obtain a Cas9 protein mutant library. The plasmid of the Cas9 protein mutant library was transformed into yeast cells to construct yeast cells containing the Cas9 mutant library; The reporter vector and the sgRNA expression vector were co-transformed into the yeast cells; Transformed yeast cells were cultured, and monoclonal colonies that could activate the reporter gene were screened. After verification, non-NGG-dependent Cas9 mutants were obtained.

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