CN110577971B - CRISPR/Sa-SauriCas9 gene editing system and application thereof - Google Patents

Abstract

The invention belongs to the technical field of gene editing, and particularly relates to CRISPR (clustered regularly interspaced short palindromic repeats)Sa‑SauriCas9A gene editing system and applications thereof. The gene editing system of the invention isSa‑SauriCas9A complex formed by the protein and the sgRNA can accurately target a DNA sequence and generate cutting, so that double-strand break damage occurs to the DNA; the gene editing is gene editing in a cell or in vitro;Sa‑SauriCas9for fusion proteins, the PAM recognition domain (PAM intervening, PI) of SaCas9 was replaced with the PAM recognition domain of saurcias 9 (saurcicas 9-PI).Sa‑SauriCas9The protein is small, is 1056 amino acids, and the identified PAM sequence is simple, theSa‑ SauriCas9The protein has an amino acid sequence shown in SEQ ID NO. 1, and the sgRNA has a nucleotide sequence shown in SEQ ID NO. 2. The invention has wide application prospect in the field of gene editing.

Description

CRISPR/Sa-SauriCas9 gene editing system and application thereof

Technical Field

The invention belongs to the technical field of gene editing, and particularly relates to a CRISPR/Sa-SauriCas9 system capable of performing gene editing in cells and related application thereof.

Background

CRISPR/Cas9 is an acquired immune system evolved by bacteria and archaea to protect against foreign virus or plasmid invasion. In a CRISPR/Cas9 system, after a CRISPR (CRISPR-derived RNA), a tracrRNA (trans-activating RNA) and a Cas9 protein form a complex, a PAM (Protospace Adjacent Motif) sequence of a target site is recognized, the CRRNA and the target DNA sequence form a complementary structure, and the Cas9 protein plays a role in cutting DNA so as to break and damage the DNA. Among them, tracrRNA and crRNA can be fused into a single-stranded guide RNA (sgRNA) by a linker sequence. When DNA breaks and damages, two major DNA damage repair mechanisms within the cell are responsible for repair: non-homologous end-joining (NHEJ) and Homologous Recombination (HR). Deletion or insertion of a base can be caused as a result of NHEJ repair, and gene knockout can be carried out; in the case of providing a homologous template, site-directed insertion of genes and precise base substitution can be performed using HR repair.

Besides basic scientific researches, the CRISPR/Cas9 also has wide clinical application prospects. When the CRISPR/Cas9 system is used for gene therapy, cas9 and sgRNA need to be introduced into a body. The most effective delivery vector for gene therapy is AAV. However, AAV virus-packaged DNA typically does not exceed 4.5kb. SpCas9 is widely used because of its simple PAM sequence (recognition of NGG) and high activity. However, the SpCas9 protein has 1367 amino acids, and the sgRNA and the promoter cannot be effectively packaged into the AAV virus, so that the clinical application of the protein is limited. To overcome this problem, several small Cas9 were invented, including SaCas9 (PAM sequence NNGRRT); st1Cas9 (PAM sequence NNAGAW); nmCas9 (PAM sequence NNNNGATT); nme2Cas9 (PAM sequence NNNNCC); cjCas9 (PAM sequence is NNRYAC), but these Cas9 or PAM sequences are complex (few DNA sequences can be targeted in genome), or the editing efficiency is low, which is difficult to be widely applied. The search for a small Cas9 protein, PAM sequence simple CRISPR/Cas9 system is hopeful to solve the above problems.

Disclosure of Invention

Aiming at the problems, the invention aims to provide a novel CRISPR/Cas9 gene editing system which is high in editing activity, small in Cas9 protein and simple in PAM sequence and application thereof.

The CRISPR/Cas9 gene editing system provided by the invention is a complex formed by a Sa-SauriCas9 protein and a sgRNA and is marked as the CRISPR/SauriCas 9 gene editing system (namely, the Sa-SauriCas9 protein which realizes gene editing under the coaction with a single guide RNA (sgRNA)); the DNA sequence can be precisely targeted, and the cutting is generated, so that the DNA is subjected to double-strand break damage; the gene editing is gene editing in a cell or in vitro; the Sa-SauriCas9 protein is small and 1053 amino acids, the identified PAM sequence is simple (NNGG), and the Sa-SauriCas9 protein has an amino acid sequence shown in SEQ ID NO. 1 or an amino acid sequence which is at least 80 percent identical to the amino acid sequence shown in SEQ ID NO. 1; the sgRNA has a nucleotide sequence shown in SEQ ID NO. 2, or a sgRNA sequence modified based on SEQ ID NO. 2.

In the present invention, the cells include eukaryotic cells and prokaryotic cells; the eukaryotic cells include mammalian cells and plant cells.

In the present invention, the mammalian cells include Chinese hamster ovary cells, baby hamster kidney cells, mouse Sertoli cells, mouse mammary tumor cells, buffalo rat liver cells, rat hepatoma cells, monkey kidney CVI line transformed by SV40, monkey kidney cells, canine kidney cells, human cervical cancer cells, human lung cells, human liver cells, HIH/3T3 cells, human U2-OS osteosarcoma cells, human A549 cells, human K562 cells, human HEK293T cells, human HCT116 cells, or human MCF-7 cells or TRI cells.

In the invention, sa-SauriCas9 in the CRISPR/Cas9 system is a fusion protein, the PI domain of SauriCas9 is replaced by the PI domain of SauriCas9, and the SauriCas9 is Staphylococcus auricularis Cas9. The Sa-SauriCas9 fusion protein and single guide RNA (sgRNA) work together to achieve gene editing.

In the invention, the SauriCas9 protein belongs to Staphylococcus aureus (Staphylococcus aureus), and the search number of the UniProt of the SauriCas9 protein is A0A2T4M4R5.

In the present invention, the Sa-SauriCas9 protein includes a Sa-SauriCas9 protein having no cleavage activity or having only a single strand cleavage activity or having a double strand cleavage activity.

In the invention, the accurate positioning DNA sequence comprises a 5' end 20bp or 21bp sequence in the sgRNA and a target DNA sequence which can form a base complementary pairing structure.

In the invention, the accurate positioning target DNA sequence comprises a Sa-SauriCas9 protein and a PAM sequence on a sgRNA complex recognition target DNA sequence.

In the invention, the PAM sequence is NNGG, and the target DNA sequence is:

NNNNNNNNNNNNNNNNNNNNNNNGG(SEQ ID NO:3)。

in the invention, the Sa-SauriCas9 protein and sgRNA complex can precisely target DNA sequences, namely the Sa-SauriCas9 protein and the sgRNA complex can recognize and combine specific DNA sequences, or other proteins fused with the Sa-SauriCas9 protein or proteins capable of specifically recognizing sgRNA are brought to the position of the target DNA.

In the present invention, the Sa-saurtias 9 protein and sgRNA complex or other proteins fused with the Sa-saurtias 9 protein or proteins specifically recognizing sgrnas can modify and regulate the targeted DNA region, including but not limited to regulation of gene transcription level, DNA methylation regulation, DNA acetylation modification, histone acetylation modification, single base converter or chromatin imaging tracking.

In the present invention, the single base converter includes, but is not limited to, conversion between bases adenine to guanine, or cytosine to thymine, or cytosine to uracil, or other bases.

The gene editing system provided by the invention has high editing activity and has obvious advantages compared with the prior Cas9.

The editing efficiency of the CRISPR/Sa-SauriCas9 system is detected by the technologies of gene synthesis, molecular cloning, cell transfection, PCR product deep sequencing, bioinformatics analysis and the like.

The CRISPR/SauriCas 9 gene editing system provided by the invention can carry out gene editing in cells, and comprises the steps of identifying and positioning target DNA through a compound of Sa-SauriCas9 protein and sgRNA, and editing the DNA; and finally, detecting the editing efficiency. The method comprises the following specific steps:

(1) Synthesizing a humanized Sa-SauriCas9 gene sequence; and cloning to an expression vector to obtain pAAV2_ Sa-SauriCas9_ ITR;

(2) Synthesizing oligonucleotide single-stranded DNA (deoxyribonucleic acid) corresponding to the sgRNA, namely Oligo-F and Oligo-R sequences, annealing and connecting to a BsaI enzyme digestion site of a plasmid pAAV2_ SauriCas9_ U6_ BsaI to obtain pAAV2_ SauriCas9-hU6-sgRNA;

(3) Delivering a vector expressing the Sa-SauriCas9 protein, sgRNA, into a cell containing a target site;

(4) And carrying out PCR amplification on the edited target site, and detecting the editing efficiency by T7EI enzyme digestion or second-generation sequencing.

In the present invention, any targeted sgRNA can be designed for a DNA sequence to be edited according to specific needs, and modifications well known in the art including, but not limited to, phosphorylation, shortening, lengthening, sulfurization, methylation, and hydroxylation can be performed on the sgRNA to some extent.

In the present invention, the CRISPR/Sa-saurtias 9 system delivered to the cell may include, but is not limited to, a plasmid expressing the Sa-saurtias 9 protein or sgRNA, a retrovirus, adenovirus, adeno-associated viral vector or RNA, or Sa-saurtias 9 protein, according to specific needs.

It will be appreciated by those skilled in the art that the base N represents any of the four bases A, T, C or G.

Further, in step (3), the delivery means includes, but is not limited to, liposomes, cationic polymers, nanoparticles, multifunctional envelope-type nanoparticles, and viral vectors.

Further, in step (3), the cells include, but are not limited to, human cells, animal cells, plant cells, bacterial cells, and fungal cells.

Further, in the step (2), the sgRNA has a nucleotide sequence shown in SEQ ID NO. 2, or a nucleotide sequence at least 80% identical to the nucleotide sequence shown in SEQ ID NO. 2, or modifications based on the nucleotide sequence, wherein the modifications include but are not limited to phosphorylation, shortening, lengthening, sulfurization or methylation.

More specifically, in one embodiment, the oligonucleotide single-stranded DNA sequences corresponding to sgrnas, i.e., oligo-F and Oligo-R, were synthesized as follows:

Oligo-F CACCGCTCGGAGATCATCATTGCG，(SEQ ID NO:4)

Oligo-R AAACCGCAATGATGATCTCCGAGC。(SEQ ID NO:5)

more specifically, in one embodiment, as will be understood by those skilled in the art, oligo-F and Oligo-R need to be annealed to become double-stranded DNA, the annealing reaction system is 1 μ L of 100 μ M Oligo-F,1 μ L of 100 μ M Oligo-R,28 μ L of water, after shaking and mixing, the mixture is placed in a PCR instrument to run an annealing program; the annealing procedure was as follows: 95 ℃ A. 5min,85 ℃ A. 1min,75 ℃ A. 1min,65 ℃ A. 1min,55 ℃ A. 1min,45 ℃ A. 1min,35 ℃ A. 1min,25 ℃ A. 1min,4 ℃ storage, cooling rate 0.3 ℃/s.

More specifically, in one embodiment, the plasmid pAAV2_ Sa-saurtias 9_ ITR needs to be linearized with BsaI restriction endonuclease (NEB).

More specifically, in one embodiment, the annealed Oligo-F and Oligo-R products are ligated to the linearized pAAV2_ Sa-SauriCas9_ ITR backbone vector by DNA ligase.

More specifically, in one embodiment, after transformation of the ligation products into competent cells, the correct clones were verified by Sanger sequencing and the plasmids were extracted for use.

More specifically, in one embodiment, the cell in step (3) is HEK293T comprising a target site having the nucleotide sequence shown in SEQ ID NO. 6.

More specifically, in one embodiment, the delivery means in step (3) is a liposome comprising

2000 or PEI.

More specifically, in a specific embodiment of the first aspect of the present invention, the template for PCR in the experimental step (4) is edited HEK293T genomic DNA.

More specifically, in one embodiment, the primer sequences amplified by PCR in step (4) are:

F1-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGCGAGAAAAGCCTTGTTT；(SEQ ID NO:7)

R1-ACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTGAACTTGTGGCCGTTTAC；(SEQ ID NO:8)

F2-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC；(SEQ ID NO:9)

R2-CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTG；(SEQ ID NO:10)

the invention also provides a CRISPR/Sa-SauriCas9 system kit for gene editing, which comprises a Sa-SauriCas9 protein or sgRNA of a target DNA sequence or a target DNA.

The invention also provides application of the CRISPR/Sa-SauriCas9 gene editing system, which comprises gene knockout, site-specific base change, site-specific insertion, regulation and control of gene transcription level, DNA methylation regulation and control, DNA acetylation modification, histone acetylation modification, single base converter or chromatin imaging tracking.

Drawings

FIG. 1 is a schematic diagram of the CRISPR/Sa-SauriCas9 gene editing system cutting targeting DNA. Wherein, the grey oval represents Sa-SauriCas9 protein, the black curved shape represents sgRNA sequence, and the deepened area in the upper chain of the genome represents PAM sequence NNGG.

FIG. 2 is a map schematic of plasmid pAAV2_ SauriCas9_ U6_ BsaI. Wherein, the recombinant human RNA comprises AAV2 ITR, CMV enhancer, CMV promoter, SV40 NLS, sa-SauriCas9, nucleoplasmin NLS, 3 xHA, bGH poly (A), human U6 promoter (hU 6), bsaI endonuclease site, sgRNA scaffold sequence and other elements.

FIG. 3 shows the result of partial next generation sequencing after editing the DNA sequence of the target site. Wherein the editing result has deletion, insertion or mismatch, and the last 4bp represents PAM sequence NNGG.

FIG. 4 shows the result of cleavage of endogenous site with T7 Endonuclease I. Wherein the arrows indicate the size of the cut fragments.

Detailed Description

The present invention will be further illustrated by the following examples, which are not intended to limit the invention in any way.

The reagents, methods and apparatus employed in the present invention are conventional in the art, except as otherwise indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available. The experimental method not specified for specific conditions is usually carried out according to conventional conditions or conditions recommended by the manufacturer.

In a specific embodiment, the CRISPR/Sa-saurii cas9 system provided by the invention is a novel gene editing system, method, kit and application thereof.

In a specific embodiment of the invention, the CRISPR/Sa-saurtias 9 system is capable of gene editing in a cell, the method comprising the steps of:

1. construction of plasmid pAAV2_ Sa-SauriCas9_ ITR

And (1) according to the retrieval number A0A2T4M4R5 of the SauriCas9 gene on the UniProt, improving the downloaded SauriCas9 protein sequence to obtain the Sa-SauriCas9 gene, wherein the amino acid sequence of the SauriCas9 gene is shown as SEQ ID NO: 1.

And (2) carrying out codon optimization on the amino acid sequence of the Sa-SauriCas9 to obtain a coding sequence of the Sa-SauriCas9 highly expressed in human cells, wherein the coding sequence is shown as SEQ ID NO: 11.

And (3) carrying out gene synthesis on the coding sequence of the obtained gene Sa-SauriCas9 in a company, and constructing the coding sequence to a pAAV2_ ITR skeleton plasmid to obtain a plasmid pAAV2_ Sa-SauriCas9_ ITR, wherein the plasmid is shown in figure 2.

2. Preparation of linearized plasmid pAAV2_ Sa-SauriCas9_ ITR

Step (1), carrying out enzyme digestion linearization on the plasmid pAAV2_ Sa-SauriCas9_ ITR by using BasI restriction enzyme, wherein an enzyme digestion system comprises: mu.g of plasmid pAAV2_ SauriCas9_ ITR, 5. Mu.L of 10 xClutSmart buffer, 1. Mu.L of the endonuclease, and water to 50. Mu.L, reacted at 37 ℃ for 1 hour.

And (2) carrying out electrophoresis on the products after enzyme digestion on a 1% agarose gel at 120V for 30 minutes.

And (3) cutting off the 7433bp DNA fragment, recovering by using a gel recovery kit according to the steps provided by the manufacturer, and finally eluting by using ultrapure water.

And (4) determining the DNA concentration of the recovered linearized plasmid pAAV2_ Sa-SauriCas9_ ITR by using NanoDrop, and reserving for later use or storing at-20 ℃ for a long time.

3. Construction of plasmid pAAV2_ Sa-SauriCas9-hU6-sgRNA

And (1) designing a sgRNA sequence.

Step (2), respectively adding corresponding sticky end sequences at two sides of a linearized plasmid pAAV2_ Sa-SauriCas9_ ITR on a sense strand and an antisense strand for the designed sgRNA sequence pair, and synthesizing two oligonucleotide single-stranded DNAs at the company, wherein the specific sequences are as follows:

Oligo-F CACCGCTCGGAGATCATCATTGCG，(SEQ ID NO:4)

Oligo-R AAACCGCAATGATGATCTCCGAGC。(SEQ ID NO:5)

and (3) annealing the oligonucleotide single-stranded DNA to obtain double-stranded DNA, wherein an annealing reaction system comprises the following steps: after shaking and mixing 1. Mu.L of 100. Mu. Mooligo-F, 1. Mu.L of 100. Mu. Mooligo-R and 28. Mu.L of water, the mixture was placed in a PCR instrument to run an annealing program: 95 ℃ 5min,85 ℃ 1min,75 ℃ 1min,65 ℃ 1min,55 ℃ 1min,45 ℃ 1min,35 ℃ 1min,25 ℃ 1min,4 ℃ storage, cooling rate 0.3 ℃/s.

And (4) connecting the annealed product with the linearized plasmid pAAV2_ Sa-SauriCas9_ ITR under the action of DNA ligase according to the steps provided by the product.

Step (5), 1. Mu.L of the ligation product was taken for chemical competent transformation, and the growing bacterial clones were subjected to Sanger sequencing validation.

And (6) connecting the sequencing verification with the correctly connected clone shake bacteria, and extracting a plasmid pAAV2_ SauriCas9-hU6-sgRNA for later use.

4. Plasmid pAAV2_ Sa-SauriCas9-hU6-sgRNA for transfecting and expressing Sa-SauriCas9 protein and sgRNA

Step (1), on day 0, according to transfection needs, the HEK293T cell line containing the sgRNA targeting site is plated in a 6-well plate, the cell density is about 30%, and the sequence of the target site is shown as SEQ ID NO. 6.

Step (2), day 1, transfection was performed in the following transfection system,

i. adding 2 mu g of plasmid pAAV2_ Sa-SauriCas9-hU6-sgRNA to be transfected into 100 mu l of Opti-MEM culture medium, and gently blowing, beating and uniformly mixing;

ii. Mixing

Flicking 2000, mixing, sucking 5 μ l, adding into 100 μ l Opti-MEM culture medium, mixing, standing at room temperature for 5min;

diluting

2000 and diluted plasmid were mixed, gently whipped and mixed, left at room temperature for 20min, and then added to the medium of cells to be transfected.

And (3) placing the cells in a 5% CO2 incubator at 37 ℃ for continuous culture.

5. Preparation of a second Generation sequencing library

And (1) collecting the HEK293T cells after editing for 3 days, and extracting the genome DNA by using a DNA kit according to the steps provided by the product.

Step (2), performing first round PCR of PCR library building, performing PCR reaction by using 2xQ5 Mastermix, wherein PCR primers are shown as SEQ ID NO:7-SEQ ID NO:8, and the reaction system is as follows:

the PCR run program was as follows:

and (3) carrying out second round PCR of PCR library building, carrying out PCR reaction by using 2xQ5 Mastermix, wherein PCR primers are shown as SEQ ID NO: 9-SEQ ID NO:10, and the reaction system is as follows:

the PCR run program was as follows:

and (4) purifying DNA fragments with the size of 366bp by using a gel recovery kit for the PCR products of the second round according to the steps provided by the manufacturer, and preparing the second generation sequencing library.

6. Analysis of the results of the second generation sequencing

Step (1), the prepared second-generation sequencing library was submitted to the company for paired-end sequencing on HiseqXTen.

Step (2) bioinformatics analysis of the next-generation sequencing results, and partial compilation results are shown in FIGS. 2 and 3.

7. Endogenous site validation

Step (1), passing the plasmid pAAV2_ Sa-SauriCas9-hU6-sgRNA for expressing Sa-SauriCas9 and sgRNA through

2000 were transfected into HEK293T cells according to the manufacturer's protocol, in which,

the sgRNA sequence is: ATAGGGTTAGGGGCCCCAGGC, (SEQ ID NO: 12)

The specific sequence of the target site is as follows: ATAGGGTTAGGGGCCCCAGGCGGG; (SEQ ID NO: 13)

Extracting cell genome DNA after 5 days of editing, and amplifying a target DNA sequence by using primers Test-F and Test-R through a 2x Q5 Master mix; wherein:

the specific sequence of Test-F is: ACGCAGTGGGTCATAGGCTC (SEQ ID NO: 14)

The specific sequence of Test-R is: ggactcaggcctcctcctcct; (SEQ ID NO: 15)

Recovering the PCR product through agarose gel and purifying DNA fragment with 509bp size;

step (4), carrying out enzyme digestion on the purified DNA fragment according to the instruction of T7 Endonuclease I, then carrying out gel running detection, wherein the result is shown in figure 4, the left side is a negative control group, and sgRNA is not generated during transfection, and no cut fragment is generated after the T7 Endonuclease I cuts the target sequence, which indicates that no editing is generated; the right panel is the experimental group, sgRNA was present during transfection, and the T7 endonucleolytic I cleaved after cleavage of the targeting sequence, indicating that editing has occurred.

SEQUENCE LISTING

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<213> Artificial sequence

<400> 8

actggagttc agacgtgtgc tcttccgatc tctgaacttg tggccgttta c 51

<210> 9

<211> 45

<212> DNA

<213> Artificial sequence

<400> 9

aatgatacgg cgaccaccga gatctacact ctttccctac acgac 45

<210> 10

<211> 52

<212> DNA

<213> Artificial sequence

<400> 10

caagcagaag acggcatacg agatcactgt gtgactggag ttcagacgtg tg 52

<210> 11

<211> 3168

<212> DNA

<213> Artificial sequence

<400> 11

atgaagcgga actacatcct gggcctggac atcggcatca ccagcgtggg ctacggcatc 60

atcgactacg agacacggga cgtgatcgat gccggcgtgc ggctgttcaa agaggccaac 120

gtggaaaaca acgagggcag gcggagcaag agaggcgcca gaaggctgaa gcggcggagg 180

cggcatagaa tccagagagt gaagaagctg ctgttcgact acaacctgct gaccgaccac 240

agcgagctga gcggcatcaa cccctacgag gccagagtga agggcctgag ccagaagctg 300

agcgaggaag agttctctgc cgccctgctg cacctggcca agagaagagg cgtgcacaac 360

gtgaacgagg tggaagagga caccggcaac gagctgtcca ccaaagagca gatcagccgg 420

aacagcaagg ccctggaaga gaaatacgtg gccgaactgc agctggaacg gctgaagaaa 480

gacggcgaag tgcggggcag catcaacaga ttcaagacca gcgactacgt gaaagaagcc 540

aaacagctgc tgaaggtgca gaaggcctac caccagctgg accagagctt catcgacacc 600

tacatcgacc tgctggaaac ccggcggacc tactatgagg gacctggcga gggcagcccc 660

ttcggctgga aggacatcaa agaatggtac gagatgctga tgggccactg cacctacttc 720

cccgaggaac tgcggagcgt gaagtacgcc tacaacgccg acctgtacaa cgccctgaac 780

gacctgaaca atctcgtgat caccagggac gagaacgaga agctggaata ttacgagaag 840

ttccagatca tcgagaacgt gttcaagcag aagaagaagc ccaccctgaa gcagatcgcc 900

aaagaaatcc tcgtgaacga agaggatatt aagggctaca gagtgaccag caccggcaag 960

cccgagttca ccaacctgaa ggtgtaccac gacatcaagg acattaccgc ccggaaagag 1020

attattgaga acgccgagct gctggatcag attgccaaga tcctgaccat ctaccagagc 1080

agcgaggaca tccaggaaga actgaccaat ctgaactccg agctgaccca ggaagagatc 1140

gagcagatct ctaatctgaa gggctatacc ggcacccaca acctgagcct gaaggccatc 1200

aacctgatcc tggacgagct gtggcacacc aacgacaacc agatcgctat cttcaaccgg 1260

ctgaagctgg tgcccaagaa ggtggacctg tcccagcaga aagagatccc caccaccctg 1320

gtggacgact tcatcctgag ccccgtcgtg aagagaagct tcatccagag catcaaagtg 1380

atcaacgcca tcatcaagaa gtacggcctg cccaacgaca tcattatcga gctggcccgc 1440

gagaagaact ccaaggacgc ccagaaaatg atcaacgaga tgcagaagcg gaacgccgcc 1500

accaacgagc ggatcgagga aatcatccgg accaccggca aagagaacgc caagtacctg 1560

atcgagaaga tcaagctgca cgacatgcag gaaggcaagt gcctgtacag cctggaagcc 1620

atccctctgg aagatctgct gaacaacccc ttcaactatg aggtggacca catcatcccc 1680

agaagcgtgt ccttcgacaa cagcttcaac aacaaggtgc tcgtgaagca ggaagaaaac 1740

agcaagaagg gcaaccggac cccattccag tacctgagca gcagcgacag caagatcagc 1800

tacgaaacct tcaagaagca catcctgaat ctggccaagg gcaagggcag aatcagcaag 1860

accaagaaag agtatctgct ggaagaacgg gacatcaaca ggttctccgt gcagaaagac 1920

ttcatcaacc ggaacctggt ggataccaga tacgccaccg ccgccctgat gaacctgctg 1980

cggagctact tcagagtgaa caacctggac gtgaaagtga agtccatcaa tggcggcttc 2040

accagctttc tgcggcggaa gtggaagttt aagaaagagc ggaacaaggg gtacaagcac 2100

cacgccgagg acgccctgat cattgccaac gccgatttca tcttcaaaga gtggaagaaa 2160

ctggacaagg ccaaaaaagt gatggaaaac cagatgttcg aggaaaagca ggccgagagc 2220

atgcccgaga tcgaaaccga gcaggagtac aaagagatct tcatcacccc ccaccagatc 2280

aagcacatta aggacttcaa ggactacaag tacagccacc gggtggacaa gaagcctaat 2340

agagagctga ttaacgacac cctgtactcc acccggaagg acgacaaggg caacaccctg 2400

atcgtgaaca atctgaacgg cctgtacgac aaggacaatg acaagctgaa aaagctgatc 2460

aacaagagcc ccgaaaagct gctgatgtac caccacgacc cccagaccta ccagaaactg 2520

aagctgatta tggaacagta cggcgacgag aagaatcccc tgtacaagta ctacgaggaa 2580

accgggaact acctgaccaa gtactccaaa aaggacaacg gccccgtgat caagaagatt 2640

aagtattacg gcaacaaact gaacgcccat ctggacatca ccgacgacta ccccaacagc 2700

agaaacaagg tcgtgaagct gtccctgaag agcttccgct tcgacatcta caagtgcgag 2760

cagggctaca agatggtgag catcggctac ctggacgtgc tgaagaagga caactactac 2820

tacatcccca aggacaagta cgaggccgag aagcagaaga agaagataaa ggagagcgac 2880

ctgttcgtgg gcagcttcta ctacaacgac ctgataatgt acgaggacga gctgttccgc 2940

gtgataggcg tgaacagcga catcaacaac ctggtggagc tgaacatggt ggacatcacc 3000

tacaaggact tctgcgaggt gaacaacgtg accggcgaga agcgcatcaa gaagaccatc 3060

ggcaagcgcg tggtgctgat agagaagtac accaccgaca tcctgggcaa cctgtacaag 3120

acccccctgc ccaagaagcc ccagctgata ttcaagcgcg gcgagctg 3168

<210> 12

<211> 21

<212> DNA

<213> Artificial sequence

<400> 12

atagggttag gggccccagg c 21

<210> 13

<211> 25

<212> DNA

<213> Artificial sequence

<400> 13

atagggttag gggccccagg ccggg 25

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence

<400> 14

acgcagtggg tcataggctc 20

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence

<400> 15

ggactcaggc ccttcctcct 20

Claims (15)

1. A CRISPR/Cas9 gene editing system is used for gene editing in cells or in vitro and is characterized in that the CRISPR/Cas9 system is a Sa-SauriCas9 protein and sgRNA complex, can accurately position a target DNA sequence and generate cutting, so that double-strand break damage occurs to DNA; the Sa-SauriCas9 protein is an amino acid sequence shown as SEQ ID NO. 1; the sgRNA is a nucleotide sequence shown in SEQ ID NO. 2.

2. The CRISPR/Cas9 gene editing system of claim 1, wherein the cells comprise eukaryotic cells and prokaryotic cells; the eukaryotic cells include mammalian cells and plant cells; the mammalian cell includes a Chinese hamster ovary cell, a baby hamster kidney cell, a mouse Sertoli cell, a mouse mammary tumor cell, a buffalo rat liver cell, a rat hepatoma cell, a monkey kidney CVI line transformed by SV40, a monkey kidney cell, a canine kidney cell, a human cervical cancer cell, a human lung cell, a human liver cell, an HIH/3T3 cell, a human U2-OS osteosarcoma cell, a human A549 cell, a human K562 cell, a human HEK293T cell, a human HCT116 cell, or a human MCF-7 cell or a TRI cell.

3. The CRISPR/Cas9 gene editing system of claim 1, wherein the precisely located targeting DNA sequence comprises a sequence of 20bp or 21bp at the 5' end of the sgRNA which can form a base complementary pairing structure with the targeting DNA sequence.

4. The CRISPR/Cas9 gene editing system of claim 1, wherein the precisely located targeting DNA sequence comprises a PAM sequence on the Sa-SauriCas9 protein and sgRNA complex recognition targeting DNA sequence.

5. The CRISPR/Cas9 gene editing system of claim 4, wherein the PAM sequence is NNGG and the targeting DNA sequence is shown in SEQ ID NO. 3.

6. The CRISPR/Cas9 gene editing system of claim 1, wherein the sgRNA can be phosphorylated, sulfurized, methylated, or hydroxylated modified.

7. The CRISPR/Cas9 gene editing system according to claim 1, wherein the Sa-SauriCas9 protein and sgRNA complex can precisely locate the target DNA sequence, which means that the Sa-SauriCas9 protein and sgRNA complex can recognize and bind to a specific DNA sequence, or that other proteins fused with the Sa-SauriCas9 protein or proteins specifically recognizing sgRNA are brought to the position of the target DNA.

8. The CRISPR/Cas9 gene editing system of claim 7, wherein the Sa-SauriCas9 protein and sgRNA complex or other proteins fused to Sa-SauriCas9 protein or proteins specifically recognizing sgrnas can modify and regulate targeted DNA regions, including regulation of gene transcription levels, single base switch or chromatin imaging tracking.

9. The CRISPR/Cas9 gene editing system of claim 8, wherein the single base switch comprises a switch between bases adenine to guanine, cytosine to thymine, cytosine to uracil, or other bases.

10. A method of gene editing for non-therapeutic purposes by the CRISPR/Cas9 gene editing system as claimed in any of claims 1 to 9 in a cell comprising editing DNA by recognition and localization of the targeted DNA by the complex of Sa-SauriCas9 protein and sgRNA; finally, detecting the editing efficiency; the method comprises the following specific steps:

(1) Synthesizing a humanized SauriCas9-PI gene sequence; and cloning to an expression vector to obtain pAAV2_ Sa-SauriCas9_ ITR;

(2) Synthesizing oligonucleotide single-stranded DNA (deoxyribonucleic acid) corresponding to the sgRNA, namely Oligo-F and Oligo-R sequences, annealing and connecting to a BsaI enzyme digestion site of a plasmid pAAV2_ SauriCas9_ U6_ BsaI to obtain pAAV2_ SauriCas9-hU6-sgRNA;

(3) Delivering a vector expressing the Sa-SauriCas9 protein, sgRNA, into a cell containing a target site;

(4) And carrying out PCR amplification on the edited target site, and detecting the editing efficiency by T7EI enzyme digestion or second-generation sequencing.

11. The method of claim 10 wherein the pAAV2_ Sa-sauri cas9-hU6-sgRNA is an adeno-associated viral backbone plasmid comprising AAV2 ITRs, CMV enhancer, CMV promoter, SV40 NLS, sa-sauri cas9, nucleoplasmin NLS, 3x HA, bGH poly (a), human U6 promoter, bsaI endonuclease site, sgRNA scaffold sequence.

12. The method according to claim 10, wherein the CRISPR/Sa-saurtias 9 system delivered to the cell comprises a plasmid, retrovirus, adenovirus, adeno-associated viral vector or RNA expressing the Sa-saurtias 9 protein or sgRNA, or a Sa-saurtias 9 protein.

13. The method of claim 10, wherein the sgRNA is synthesized with corresponding oligonucleotide single-stranded DNA sequences, i.e., oligo-F and Oligo-R sequences are shown in SEQ ID NO 4 and SEQ ID NO 5;

the target site of the cell in the step (3) has a nucleotide sequence shown by SEQ ID NO. 6;

the template of PCR in the step (4) is edited DNA; the primer sequences for PCR amplification were: SEQ ID NO. 7, 8, 9, 10.

14. A kit based on the CRISPR/Cas9 gene editing system of any of claims 1 to 9, comprising a Sa-saurtia 9 protein and sgrnas targeting the DNA sequence.

15. Use of the CRISPR/Cas9 gene editing system according to any of claims 1 to 9 for non-therapeutic purposes including gene knock-out, site-directed base change, site-directed insertion, regulation of gene transcription level, single base switch or chromatin imaging follow-up.

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