阅读说明：本技术 CRISPR/Sa-ShaCas9基因编辑系统及其应用 (CRISPR/Sa-ShaCas9 gene editing system and application thereof ) 是由 王永明 胡子英 王大奇 王帅 于 2019-08-08 设计创作，主要内容包括：本发明属于基因编辑技术领域,具体为一种CRISPR/<I>Sa-ShaCas9</I>基因编辑系统以及其应用。本发明基因编辑系统为<I>Sa-ShaCas9</I>蛋白与sgRNA形成的复合体,能精确靶向DNA序列,并产生切割,使DNA发生双链断裂损伤；所述基因编辑为在细胞中或体外进行基因编辑；<I>Sa-ShaCas9</I>为融合蛋白,把SaCas9的PAM识别结构域(PAM interacting,PI)替换为ShaCas9的PAM识别结构域(ShaCas9-PI),<I>Sa-ShaCas9</I>蛋白小,为1055个氨基酸,识别的PAM序列简单,所述<I>Sa-ShaCas9</I>蛋白具有SEQ ID NO:1所示的氨基酸序列,所述sgRNA具有SEQ ID NO:2所示的核苷酸序列。本发明在基因编辑领域中具有广泛的应用前景。(The invention belongs to the technical field of gene editing, and particularly relates to CRISPR (clustered regularly interspaced short palindromic repeats) Sa‑ShaCas9 A gene editing system and applications thereof. The gene editing system of the invention is Sa‑ShaCas9 A 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‑ShaCas9 For fusion proteins, the PAM recognition domain (PAM interaction, PI) of SaCas9 was replaced with the PAM recognition domain of ShaCas9 (ShaCas 9-PI), Sa‑ShaCas9 The protein is small, is 1055 amino acids and recognizesThe PAM sequence of (a) is simple Sa‑ShaCas9 The 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.)

1. A CRISPR/Cas9 gene editing system for gene editing in cells or in vitro, characterized in that the CRISPR/Cas9 system isSa-ShaCas9The protein and sgRNA complex can accurately position a target DNA sequence and generate cutting, so that double-strand break damage occurs to DNA; the above-mentionedSa-ShaCas9The 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.

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 liver tumor 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 according to claim 1, wherein the CRISPR/Cas9 gene editing systemSa-ShaCas9proteins comprising no cleavage activity or having only single strand cleavage activity or having double strand cleavage activitySa-ShaCas9a protein.

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

5. The CRISPR/Cas9 gene editing system according to claim 1, wherein the precisely located targeting DNA sequence comprisesSa-ShaCas9The protein and sgRNA complex recognizes a PAM sequence on the target DNA sequence.

6. The CRISPR/Cas9 gene editing system according to claim 5, wherein the PAM sequence is NNGRM and the targeting DNA sequence is shown in SEQ ID NO 3.

7. The CRISPR/Cas9 gene editing system according to claim 1, wherein the sgRNA can be phosphorylated, shortened, lengthened, sulfurized, methylated, or hydroxylated modified.

8. The CRISPR/Cas9 gene editing system according to claim 1, wherein the CRISPR/Cas9 gene editing systemSa-ShaCas9Protein and sgRNA complex can precisely target DNA sequenceSa-ShaCas9The complex of protein and sgRNA can recognize and bind to a specific DNA sequence, or to be bound toSa-ShaCas9Other proteins of the protein fusion or proteins that specifically recognize the sgRNA are brought into position to target the DNA.

9. The CRISPR/Cas9 gene editing system according to claim 8, wherein the CRISPR/Cas9 gene editing systemSa-ShaCas9Protein and sgRNA complexes or withSa-ShaCas9Other proteins of the protein fusion or proteins that specifically recognize sgrnas can be directed to the targeted DNA regionDomains are modified and regulated, including regulation of gene transcription levels, DNA methylation regulation, DNA acetylation modification, histone acetylation modification, single base converters, or chromatin imaging tracking.

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

11. CRISPR/according to one of claims 1 to 10Sa-ShaCas9A method for gene editing in a cell by a gene editing system comprisingSa-ShaCas9Identifying and positioning the target DNA by the compound of the protein and the sgRNA, and editing the DNA; finally, detecting the editing efficiency; the method comprises the following specific steps:

(1) Synthetic humanizationShaCas9-PIA gene sequence; and cloning the gene into an expression vector to obtain pAAV2 \ uSa- ShaCas9_ITR；

(2) Single-stranded oligonucleotides DNA corresponding to sgRNA, i.e., Oligo-F and Oligo-R sequences, were synthesized, annealed and ligated to plasmid pAAV2 \\ uSa-ShaCas9BsaI cleavage site of U6 BsaI to obtain pAAV 2USa-ShaCas9-hU6-sgRNA；

(3) Will expressSa-ShaCas9The protein, vector of sgRNA, is delivered into a cell containing the target site;

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

12. The method of claim 11 wherein said pAAV2 \uSa-ShaCas9-hU6-sgRNA is an adeno-associated virus backbone plasmid comprising AAV2ITR, CMV enhancer, CMV promoter, SV40NLS, Sa-ShaCas9, nucleoplasmin NLS, 3 xha, bGH poly (a), human U6 promoter, BsaI endonuclease site, sgRNA scaffold sequence.

13. the method of claim 11, wherein the step of removing the metal oxide layer comprises removing the metal oxide layer from the metal oxide layerIn that, the CRISPR delivered to the cellSa-ShaCas9The system includes an expressionSa-ShaCas9plasmids, retroviruses, adenoviruses, adeno-associated viral vectors or RNAs or of proteins or sgRNAsSa-ShaCas9a protein.

14. The method of claim 11, wherein the sequences of single-stranded oligonucleotides corresponding to sgrnas, i.e., Oligo-F and Oligo-R, are shown in SEQ ID NOs 4 and 5, are synthesized.

15. the method according to claim 11, wherein the target site of the cell in step (3) has the nucleotide sequence shown as SEQ ID NO 6.

16. the method according to claim 11, wherein the template for PCR in step (4) is edited DNA; the primer sequences for PCR amplification are: SEQ ID NO7, SEQ ID NO8, SEQ ID NO9, SEQ ID NO 10.

17. CRISPR/according to one of claims 1 to 10Sa-ShaCas9A kit for a gene editing system, the kit comprisingSa-ShaCas9sgRNA or targeting DNA of a protein or targeting DNA sequence.

18. CRISPR/according to one of claims 1 to 10Sa-ShaCas9Applications of gene editing systems, including gene knockout, site-directed base alteration, site-directed insertion, regulation of gene transcription levels, regulation of DNA methylation, DNA acetylation modification, histone acetylation modification, single base converters, or chromatin imaging tracking.

Technical Field

the invention belongs to the technical field of gene editing, and particularly relates to a CRISPR/Sa-ShaCas9 system capable of performing gene editing in cells and related application thereof.

Background

CRISPR/Cas9 is an acquired immune system that bacteria and archaea have evolved to protect against foreign virus or plasmid invasion. In the CRISPR/Cas9 system, after a complex is formed by crRNA (CRISPR-derived RNA), tracrRNA (trans-activating RNA) and Cas9 protein, a pam (promoter adjacentmotif) sequence for identifying a target site can form a complementary structure with a target DNA sequence, and Cas9 protein plays a role in cutting DNA, so that the DNA is subjected to breaking damage. Wherein, tracrRNA and crRNA can be fused into single-stranded guide RNA (sgRNA) through a connecting 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 research, the CRISPR/Cas9 also has wide clinical application prospect. 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 4.5 kb. SpCas9 has been 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 editing efficiency is low, and wide application is difficult. The search for a small Cas9 protein, PAM sequence simple CRISPR/Cas9 system is hopeful to solve the above problems.

Disclosure of Invention

In view of the above problems, the present invention aims to provide a novel gene editing system of CRISPR/Cas9 with high editing activity, small Cas9 protein and simple PAM sequence, and applications thereof.

The CRISPR/Cas9 gene editing system provided by the invention is a complex formed by a Sa-ShaCas9 protein and a sgRNA, and is marked as a CRISPR/Sa-ShaCas9 gene editing system (namely, the Sa-ShaCas9 protein which realizes gene editing under the coaction with a shingle 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 carried out in cells or in vitro; the Sa-ShaCas9 protein is small, is 1053 amino acids, and has a simple identified PAM sequence (NNGRM), and the Sa-ShaCas9 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 liver tumor 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, the Sa-ShaCas9 in the CRISPR/Cas9 system is a fusion protein, the PI domain of SaCas9 is replaced by the PI domain of ShaCas9, and the ShaCas9 is Staphylocccus haemolyticus Cas 9. The Sa-ShaCas9 fusion protein and single guide RNA (sgRNA) act together to achieve gene editing.

in the invention, the ShaCas9 protein belongs to Staphylococcus haemolyticus (Staphylococcus haemolyticus), and the search number of the Uniprot of the ShaCas9 protein is A0A2T4SLN 6.

In the invention, the Sa-ShaCas9 protein comprises a Sa-ShaCas9 protein which has no cleavage activity or only single-strand cleavage activity or double-strand cleavage activity.

In the invention, the precise positioning DNA sequence comprises a sequence of 20bp or 21bp at the 5' end of 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-ShaCas9 protein and a PAM sequence on a sgRNA complex recognition target DNA sequence.

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

NNNNNNNNNNNNNNNNNNNNNNNGRM(SEQ ID NO:3)。

In the invention, the Sa-ShaCas9 protein and sgRNA complex can precisely target DNA sequences, namely that the Sa-ShaCas9 protein and the sgRNA complex can recognize and bind to specific DNA sequences, or other proteins fused with the Sa-ShaCas9 protein or proteins specifically recognizing sgRNA are brought to the position of the target DNA.

In the present invention, the Sa-ShaCas9 protein and sgRNA complex or other proteins fused to the Sa-ShaCas9 protein or proteins specifically recognizing sgrnas may 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 transducer 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 Cas 9.

The editing efficiency of the CRISPR/Sa-ShaCas9 system is detected by the techniques of gene synthesis, molecular cloning, cell transfection, PCR product deep sequencing, bioinformatics analysis and the like.

The CRISPR/Sa-ShaCas9 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-ShaCas9 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-ShaCas9 gene sequence; and cloning to an expression vector to obtain pAAV2_ Sa-ShaCas9_ ITR;

(2) Synthesizing oligonucleotide single-stranded DNA (namely Oligo-F and Oligo-R sequences) corresponding to the sgRNA, annealing, and connecting to a BsaI enzyme cutting site of a plasmid pAAV2_ Sa-ShaCas9_ U6_ BsaI to obtain pAAV2_ Sa-ShaCas9-hU 6-sgRNA;

(3) Delivering a vector expressing the Sa-ShaCas9 protein, sgRNA, into a cell containing a target site;

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

In the present invention, according to specific needs, any targeted sgRNA can be designed for a DNA sequence to be edited, 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-ShaCas9 system that can be delivered to cells includes, but is not limited to, a plasmid expressing a Sa-ShaCas9 protein or sgRNA, a retrovirus, an adenovirus, an adeno-associated viral vector or RNA, or a Sa-ShaCas9 protein, according to specific needs.

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

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

More specifically, in one embodiment, the plasmid pAAV2_ Sa-ShaCas9_ ITR requires linearization 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-ShaCas9_ ITR backbone vector using 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 stepsThe delivery means in step (3) is a liposome comprising2000 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-ShaCas9 system kit for gene editing, which comprises a Sa-ShaCas9 protein or sgRNA of a target DNA sequence or a target DNA.

the invention also provides application of the CRISPR/Sa-ShaCas9 gene editing system, which comprises gene knockout, site-specific base change, site-specific insertion, gene transcription level regulation, DNA methylation regulation, DNA acetylation modification, histone acetylation modification, single base converter or chromatin imaging tracking.

Drawings

FIG. 1 is a schematic diagram of CRISPR/Sa-ShaCas9 gene editing system cutting targeting DNA. Wherein, the grey oval represents the Sa-ShaCas9 protein, the black curved represents the sgRNA sequence, and the darkened region in the upper chain of the genome represents the PAM sequence NNGG.

FIG. 2 is a map schematic of plasmid pAAV2_ Sa-ShaCas9_ U6_ BsaI. Wherein, the gene comprises AAV2ITR, CMV enhancer, CMV promoter, SV40NLS, Sa-ShaCas9, nucleoplasmin NLS, 3 xHA, bGH poly (A), human U6 promoter (hU6), BsaI endonuclease site, sgRNA scaffold sequence and other elements.

FIG. 3 shows the result of partial next generation sequencing after the DNA sequence of the target site has been edited. Wherein the editing result has deletion, insertion or mismatching, and the last 5bp represents a PAM sequence NNGRM.

FIG. 4 shows the cleavage of endogenous site with T7Endonuclease 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.

Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, the reagents and materials used in the following examples are commercially available. The experimental procedures not specified for the specific conditions are usually carried out according to the conventional conditions or the conditions recommended by the manufacturers.

In a specific embodiment, the CRISPR/Sa-ShaCas9 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-ShaCas9 system is capable of gene editing in a cell, the method comprising the steps of:

1. Construction of plasmid pAAV2_ Sa-ShaCas9_ ITR

Step (1), downloading the amino acid sequence of Sa-ShaCas9 gene according to the retrieval number A0A2T4SLN6 of the gene on UniProt, as shown in SEQ ID NO: 1.

And (2) carrying out codon optimization on the amino acid sequence of the Sa-ShaCas9 to obtain a coding sequence highly expressed in human cells by the Sa-ShaCas9, which is shown as SEQ ID NO: 11.

Step (3), the coding sequence of the obtained gene Sa-ShaCas9 is subjected to gene synthesis in a company and is constructed on a pAAV2_ ITR skeleton plasmid to obtain a plasmid pAAV2_ Sa-ShaCas9_ ITR, as shown in FIG. 2.

2. Preparation of linearized plasmid pAAV2_ Sa-ShaCas9_ ITR

Step (1), carrying out enzyme digestion linearization on the plasmid pAAV2_ Sa-ShaCas9_ ITR by using BasI restriction enzyme, wherein an enzyme digestion system comprises: mu.g of plasmid pAAV2_ Sa-ShaCas9_ ITR, 5. mu.L of 10xCutSmart buffer, 1. mu.L of the endonuclease, water to 50. mu.L, reacted at 37 ℃ for 1 hour.

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

And (3) cutting off the 7430bp DNA fragment, recovering by using a glue 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-ShaCas9_ ITR by using NanoDrop, and reserving or storing at-20 ℃ for long-term storage.

3. Construction of plasmid pAAV2_ Sa-ShaCas9-hU6-sgRNA

And (1) designing a sgRNA sequence.

Step (2), adding linearized plasmids pAAV2 \ to the sense strand and antisense strand of the designed sgRNA sequence pair

The corresponding cohesive end sequences of Sa-ShaCas9_ ITR both sides, and two oligonucleotide single-stranded DNAs were synthesized at the company, the specific sequences are:

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-ShaCas9_ 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) carrying out sequencing verification on the correctly connected clone shake bacteria, and extracting a plasmid pAAV2_ Sa-ShaCas9-hU6-sgRNA for later use.

4. Plasmid pAAV2_ Sa-ShaCas9-hU6-sgRNA for transfecting and expressing Sa-ShaCas9 protein and sgRNA

Step (1), on day 0, according to transfection requirements, 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 μ g of plasmid pAAV2_ Sa-ShaCas9-hU6-sgRNA to be transfected into 100 μ l of Opti-MEM culture medium, and gently blowing, beating and mixing;

ii. mixing2000 flick and mix evenly, suck 5 mul and add to 100 mul Opti-MEM culture medium, mix evenly gently, stand 5min at room temperature;

Will dilute2000 and diluted plasmid, gently pumping and mixing, standing at room temperature for 20min, and adding into the culture medium of cells to be transfected.

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

5. Preparation of a second Generation sequencing library

And (1) collecting HEK293T cells after editing for 3 days, and extracting genomic 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 2xQ5Mastermix, 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 2xQ5Mastermix, 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 PCR products of the second round according to the steps provided by the manufacturer, and finishing the preparation of 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 plasmid pAAV2_ Sa-ShaCas9-hU6-sgRNA expressing Sa-ShaCas9 and sgRNA through2000 were transfected into HEK293T cells according to the manufacturer's protocol, in which,

the sgRNA sequence is: AGTGAGGGGAACAAAGTGGAC (SEQ ID NO:12)

The specific sequence of the target site is as follows: AGTGAGGGGAACAAAGTGGACATGGC, respectively; (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 2x Q5Master mix; wherein:

The specific sequence of Test-F is: GCTGCTTTCCTGCTGTCTTC (SEQ ID NO:14)

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

recovering the PCR product through agarose gel, and purifying the DNA fragment with the size of 284 bp;

Step (4), carrying out enzyme digestion on the purified DNA fragment according to the instruction of T7Endonuclease I, then carrying out gel running detection, wherein the result is shown in figure 4, the left side is a negative control group, and no sgRNA is generated during transfection, and T7Endonuclease I cuts a targeting sequence and then has no cut fragment, which indicates that no editing is generated; the right panel shows the experimental group, with sgRNA at transfection, and T7 endonucleoclean I cleaved after cleavage of the targeting sequence to show that editing has occurred.

Sequence listing

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<120> CRISPR/Sa-ShaCas9 gene editing system and application thereof

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Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile Ala

340 345 350

Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu Leu

355 360 365

Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile Ser

370 375 380

Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala Ile

385 390 395 400

Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile Ala

405 410 415

Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser Gln

420 425 430

Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser Pro

435 440 445

Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala Ile

450 455 460

Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala Arg

465 470 475 480

Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln Lys

485 490 495

Arg Asn Ala Ala Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr Thr

500 505 510

Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His Asp

515 520 525

Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu Glu

530 535 540

Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile Pro

545 550 555 560

Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val Lys

565 570 575

Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr Leu

580 585 590

Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His Ile

595 600 605

Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys Glu

610 615 620

Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys Asp

625 630 635 640

Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Ala Ala Leu

645 650 655

Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val Lys

660 665 670

Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys Trp

675 680 685

Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu Asp

690 695 700

Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys Lys

705 710 715 720

Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu Lys

725 730 735

Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys Glu

740 745 750

Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys Asp

755 760 765

Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu Ile

770 775 780

Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr Leu

785 790 795 800

Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys Leu

805 810 815

Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His His

820 825 830

Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr Gly

835 840 845

Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn Tyr

850 855 860

Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys Ile

865 870 875 880

Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp Asp

885 890 895

Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Ser Tyr

900 905 910

Arg Phe Asp Val Tyr Leu Thr Asp Lys Gly Tyr Lys Phe Val Ser Ile

915 920 925

Thr Tyr Leu Asp Val Leu Lys Lys Glu Asn Tyr Tyr Tyr Ile Ser Glu

930 935 940

Ala Lys Tyr Asp Lys Leu Lys Leu Asn Lys Gly Ile Asp Asp Lys Ala

945 950 955 960

Lys Phe Ile Gly Ser Phe Tyr Tyr Asn Asp Leu Ile Glu Leu Asp Gly

965 970 975

Glu Val Tyr Thr Val Ile Gly Val Asn Asn Asp Lys Asn Asn Val Ile

980 985 990

Glu Leu Asn Leu Pro Glu Ile Arg Tyr Lys Glu Tyr Cys Glu Ile Asn

995 1000 1005

Asn Ile Lys Gly Ser Gly Arg Leu Arg Ile Thr Ile Gly Lys Lys

1010 1015 1020

Val Asn Ser Ile Arg Lys Leu Ser Thr Asp Val Leu Gly Asn Arg

1025 1030 1035

Tyr Tyr Gln Ser Phe Ala Lys Lys Pro Gln Leu Val Phe Lys Lys

1040 1045 1050

Gly Ile

1055

<210> 2

<211> 101

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(20)

<223> n is a, c, g, or u

<400> 2

nnnnnnnnnn nnnnnnnnnn guuuuaguac ucuggaaaca gaaucuacua aaacaaggca 60

aaaugccgug uuuaucucgu caacuuguug gcgagauuuu u 101

<210> 3

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(23)

<223> n is a, c, g, or t

<400> 3

nnnnnnnnnn nnnnnnnnnn nnngrm 26

<210> 4

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

caccgctcgg agatcatcat tgcg 24

<210> 5

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

aaaccgcaat gatgatctcc gagc 24

<210> 6

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (21)..(25)

<223> n is a, c, g, or t

<400> 6

gctcggagat catcattgcg nnnnn 25

<210> 7

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (34)..(37)

<223> n is a, c, g, or t

<400> 7

acactctttc cctacacgac gctcttccga tctnnnngcg agaaaagcct tgttt 55

<210> 8

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

actggagttc agacgtgtgc tcttccgatc tctgaacttg tggccgttta c 51

<210> 9

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aatgatacgg cgaccaccga gatctacact ctttccctac acgac 45

<210> 10

<211> 52

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

caagcagaag acggcatacg agatcactgt gtgactggag ttcagacgtg tg 52

<210> 11

<211> 3165

<212> DNA

<213> Artificial Sequence (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 agctaccgct tcgacgtgta cctgaccgac 2760

aagggctaca agttcgtgag catcacctac ctggacgtgc tgaagaagga gaactactac 2820

tacatcagcg aggccaagta cgacaagctg aagctgaaca agggcatcga cgacaaggcc 2880

aagttcatcg gcagcttcta ctacaacgac ctgatagagc tggacggcga ggtgtacacc 2940

gtgataggcg tgaacaacga caagaacaac gtgatagagc tgaacctgcc cgagatacgc 3000

tacaaggagt actgcgagat aaacaacatc aagggcagcg gcaggctgcg catcaccatc 3060

ggcaagaagg tgaacagcat ccgcaagctg agcaccgacg tgctgggcaa ccgctactac 3120

cagagcttcg ccaagaagcc ccagctggtg ttcaagaagg gcatc 3165

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

agtgagggga acaaagtgga c 21

<210> 13

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

agtgagggga acaaagtgga catggc 26

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gctgctttcc tgctgtcttc 20

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

aaggaggtct ctgtctgtgc 20