Method for realizing gene knockout in porcine fetal fibroblast by using CRISPR/Cas9 system

文档序号：1388838 发布日期：2020-08-18 浏览：9次中文

阅读说明：本技术 一种利用CRISPR/Cas9系统在猪胎儿成纤维细胞中实现基因敲除的方法 (Method for realizing gene knockout in porcine fetal fibroblast by using CRISPR/Cas9 system ) 是由姜爱文刘红林陶景丽李荣阳张轩于 2020-04-13 设计创作，主要内容包括：本发明公开了一种利用CRISPR/Cas9系统在猪胎儿成纤维细胞中实现基因敲除的方法,包括下述步骤：构建Cas9/sgRNA真核表达载体；构建RGS-CR双荧光代替性报告载体；将Cas9/sgRNA真核表达载体和RGS-CR报告载体共转至293T细胞中,筛选最高效的sgRNA序列；将筛选出的Cas9/sgRNA质粒电转至猪胎儿成纤维细胞中,实现敲除。本发明的方法操作简单,可在猪体细胞上可实现高效的基因编辑。(The invention discloses a method for realizing gene knockout in porcine fetal fibroblasts by using a CRISPR/Cas9 system, which comprises the following steps: constructing a Cas9/sgRNA eukaryotic expression vector; constructing an RGS-CR dual-fluorescence alternative report vector; transferring a Cas9/sgRNA eukaryotic expression vector and an RGS-CR report vector into a 293T cell together, and screening the most efficient sgRNA sequence; and electrically transferring the screened Cas9/sgRNA plasmid into a porcine fetal fibroblast to realize knockout. The method of the invention is simple to operate, and can realize high-efficiency gene editing on the somatic cells of the pigs.)

1. A method for achieving gene knockout in porcine fetal fibroblasts using the CRISPR/Cas9 system, comprising the steps of:

(1) constructing a Cas9/sgRNA eukaryotic expression vector;

(2) constructing an RGS-CR dual-fluorescence alternative report vector;

(3) co-transferring the Cas9/sgRNA eukaryotic expression vector constructed in the step (1) and the RGS-CR double-fluorescence alternative report vector constructed in the step (2) into 293T cells, and screening the most efficient sgRNA sequence;

(4) and electrically transferring the screened Cas9/sgRNA eukaryotic expression vector to a porcine fetal fibroblast to realize knockout.

2. The method of claim 1, wherein: the specific process for constructing the Cas9/sgRNA eukaryotic expression vector in the step (1) comprises the following steps:

(a) designing a sgRNA target sequence according to a target gene, and annealing the sgRNA target sequence with a viscous terminal sequence of a restriction enzyme BbsI and a complementary sequence with the viscous terminal sequence of the restriction enzyme BbsI to form a DNA double strand;

(b) and (b) digesting the plasmid px459v2.0-cas9 by Bbs I, recovering a plasmid framework, and cloning the DNA double strand obtained in the step (a) into the plasmid framework px459v2.0-cas9 to obtain px 459-sgRNA.

3. The method of claim 1, wherein: step (2) the specific process for constructing the RGS-CR dual-fluorescent alternative reporter vector comprises the following steps:

(a) designing an RGS-CR report carrier sequence of an sgRNA target sequence according to a target gene, and annealing the RGS-CR report carrier sequence with a viscous end sequence of restriction enzyme EcoRI and a complementary sequence with a viscous end sequence of restriction enzyme BamHI to form a DNA double strand;

(b) and (b) carrying out double enzyme digestion on the RGS-CR plasmid by using EcoRI and BamHI, recovering a plasmid framework, and cloning the DNA double strand obtained in the step (a) into the RGS-CR plasmid framework to obtain RGS-sgRNA.

4. The method of claim 1, wherein: the specific process of the step (3) comprises the following steps:

(a) transferring the 293T cell to a cell culture vessel, and performing transfection when the confluence degree meets the requirement;

(b) after cotransformation according to the Lipofectamine3000 instructions, fluorescence was observed 48h after transfection.

5. The method of claim 1, wherein: the specific process of the step (4) comprises the following steps:

(a) digesting the PEF transferred to the third generation by using Tryple, and then collecting the PEF into a centrifugal tube for centrifugation; adding the prepared non-antibiotic culture medium into a cell culture plate, and putting the cell culture plate into an incubator in advance for preheating;

(b) adding buffer E into the electric rotating glass tube, and inserting the pipette seat;

(c) washing the centrifuged PEF once by using DPBS, centrifuging again and discarding the DPBS; resuspending the cell pellet with Buffer R and adding Cas9/sgRNA eukaryotic expression vector at 1500V, 3 times, 10 ms;

(d) and placing the cells after electrotransformation into a cell culture plate for culture so as to realize knockout.

6. The method according to claim 1 or 2, characterized in that: the target gene targeted by gene knockout was ROSA26, and the sgRNA target sequence designed for the target gene was 5'-AGAGAAGAGGCTGTGCTCTG-3' (SEQ ID No. 2).

7. A method according to claim 1 or 3, characterized in that: the objective gene for gene knockout is ROSA26, and the RGS-CR report vector sequence of the sgRNA target sequence designed for the objective gene is 5 '-AGAGAAGAGGCTGTGCTCTGGGG(PAM) -3' (SEQ ID No. 8).

8. The method according to claim 2, wherein the annealing to form the DNA double strand in step (a) is performed by dissolving the sgRNA target sequence having the cohesive end sequence of the restriction enzyme BbsI and the complementary sequence having the cohesive end sequence of the restriction enzyme BbsI in 1uL each, and adding 1uL 10 × T4ligase buffer, 1uL T4PNK, 6uLdH₂O, mixing uniformly; heating at 37 deg.C for 30min, heating at 95 deg.C for 5min, gradient cooling to 25 deg.C, incubating at 25 deg.C for 5min, incubating at 4 deg.C for 5min, and storing at-20 deg.C for use.

9. The method according to claim 3, wherein the annealing to form the double-stranded DNA in step (a) is carried out by dissolving the RGS-CR reporter vector sequence having the cohesive end sequence of restriction enzyme EcoRI and the complementary sequence having the cohesive end sequence of restriction enzyme BamHI to 1uL each, and adding 1uL 10 × T4ligase buffer, 1uL T4PNK, 6uL dH₂O, mixing uniformly; heating at 37 deg.C for 30min, heating at 95 deg.C for 5min, gradient cooling to 25 deg.C, incubating at 25 deg.C for 5min, incubating at 4 deg.C for 5min, and storing at-20 deg.C for use.

10. The method of claim 5, wherein: the specific process of the step (4) is as follows:

(a) digesting the PEF transferred to the third generation for 3min by using Tryple, collecting the PEF into a 15mL centrifuge tube, and centrifuging the PEF at 1000rpm for 5 min; adding the prepared non-antibiotic culture medium into a 6-hole plate, wherein each hole is 2mL, and putting the plate into an incubator in advance for preheating;

(b) adding 3mL of buffer E into the electrotransfer glass tube, and inserting the electrotransfer glass tube into a pipettor seat;

(c) after PEF centrifugation, cleaning once by using DPBS, and discarding the DPBS after 5min centrifugation; resuspending the cell pellet with Buffer R, and adding 2ug Cas9/sgRNA eukaryotic expression vector at 1500V for 3 times of 10 ms;

(d) and placing the cells after the electrotransformation into a 6-hole plate, and culturing for 72h to realize knockout.

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a method for realizing gene knockout in porcine fetal fibroblasts by using a CRISPR/Cas9 system.

Background

The CRISPR-Cas system was first discovered in the bacterial and archaeal genome and is the autoimmune protective mechanism for bacteria and archaea. Among them, CRISPR performs a recognition function, and Cas protein performs a cleavage function. CRISPR clusters are composed of a Leader (Leader), multiple short and highly conserved Repeat regions (Repeat), and multiple spacers (Spacer). The Spacer region is composed of captured exogenous DNA, is similar to immunological memory, and can be recognized by a bacterial organism when the exogenous DNA containing the same sequence invades, and is cut to silence the expression of the exogenous DNA, so that the aim of protecting the safety of the organism is fulfilled.

There are 3 types of CRISPR-Cas systems, and in type 2 CRISPR-Cas system, Cas9 contains two unique active sites, RuvC and HNH, that play a role in crRNA maturation and double-stranded DNA cleavage. In addition, while the pre-crRNA is transcribed, a transactivating crRNA (tracrrna) complementary to its repeat is also transcribed and stimulates Cas9 and double-stranded RNA-specific RNaseIII nucleases to process the pre-crRNA. After maturation, the crRNA, tracrRNA and Cas9 form a complex that recognizes and binds to the crRNA complementary sequence, then unzips the DNA double strand to form an R-loop, allowing the crRNA to hybridize to the complementary strand, leaving the other strand in a free single-stranded state, then cleaves the crRNA complementary DNA strand by the HNH active site in Cas9, the RuvC active site cleaves the non-complementary strand, and finally introduces a DNA Double Strand Break (DSB).

Since 2013, researchers have introduced this system in a number of articles published in well-known journals including "science" and "nature biotechnology", and have succeeded in using this system to achieve precise genetic modification in species such as humans, mice, zebrafish, etc.

Disclosure of Invention

The invention aims to disclose a method for realizing gene knockout in porcine fetal fibroblasts by using a CRISPR/Cas9 system.

The purpose of the invention is realized by the following technical scheme:

a method of effecting gene knockout in porcine fetal fibroblasts using the CRISPR/Cas9 system, the method comprising the steps of:

(1) constructing a Cas9/sgRNA eukaryotic expression vector;

(2) constructing an RGS-CR dual-fluorescence alternative report vector;

(4) and electrically transferring the screened Cas9/sgRNA eukaryotic expression vector to a porcine fetal fibroblast to realize knockout.

As a preferred technical scheme: the specific process for constructing the Cas9/sgRNA eukaryotic expression vector in the step (1) comprises the following steps:

(c) And transforming the ligation product into DH5a, selecting a single clone, expanding and culturing, and then sequencing and identifying.

As a preferred technical scheme: step (2) the specific process for constructing the RGS-CR dual-fluorescent alternative reporter vector comprises the following steps:

(c) And transforming the ligation product into DH5a, selecting a single clone, expanding and culturing, and then sequencing and identifying.

As a preferred technical scheme: the specific process of the step (3) comprises the following steps:

(a) transferring the 293T cell to a cell culture vessel, and performing transfection when the confluence degree meets the requirement; in detail, 293T cells were transferred to 5 6cm dishes and transfected when the confluence reached 90%.

(b) After cotransformation according to the Lipofectamine3000 instructions, fluorescence was observed 48h after transfection.

As a preferred technical scheme: the specific process of the step (4) comprises the following steps:

(b) adding buffer E into the electric rotating glass tube, and inserting the pipette seat;

(d) and placing the cells after electrotransformation into a cell culture plate for culture so as to realize knockout.

Further preferred is: the gene targeted for gene knock-out is ROSA26, but is not limited thereto. The sgRNA target sequence designed for this gene of interest is 5'-AGAGAAGAGGCTGTGCTCTG-3' (SEQ ID No. 2).

The sgRNA target sequence with the sticky end sequence of the restriction enzyme BbsI was 5'-CACCGAGAGAAGAGGCTGTGCTCTG-3' (SEQ ID No. 5); the complementary sequence with the cohesive end sequence of the restriction enzyme BbsI is 5'-AAACCAGAGCACAGCCTCTTCTCTC-3' (SEQ ID No.6)

The RGS-CR report vector sequence of the sgRNA target sequence designed aiming at the target gene is 5 '-AGAGAAGAGGCTGTGCTCTGGGG(PAM) -3' (SEQ ID No. 8).

RGS-CR reporter vector with restriction enzyme EcoRI cohesive end sequenceThe sequence is 5' -AATTCAGAGAAGAGGCTGTGCTCTGGGG(PAM)-3 ' (SEQ ID No.11) with the complement of the cohesive end sequence of the restriction enzyme BamHI 5'-GATCCCCCAGAGCACAGCCTCTTCTCTG-3' (SEQ ID No. 12).

It is further preferred that annealing to form a DNA double strand in step (1) is carried out by dissolving the sgRNA target sequence having the cohesive end sequence of the restriction enzyme BbsI and the complementary sequence having the cohesive end sequence of the restriction enzyme BbsI in 1uL each, and adding 1uL 10 × T4ligase buffer, 1uL T4PNK, 6uL dH₂O, mixing uniformly; heating at 37 deg.C for 30min, heating at 95 deg.C for 5min, gradient cooling to 25 deg.C, incubating at 25 deg.C for 5min, incubating at 4 deg.C for 5min, and storing at-20 deg.C for use.

It is further preferred that the annealing to form a double-stranded DNA in the step (2) is carried out by dissolving the RGS-CR reporter vector sequence having the cohesive end sequence of restriction enzyme EcoRI and the complementary sequence having the cohesive end sequence of restriction enzyme BamHI in 1uL each, and adding 1uL 10 × T4ligase buffer, 1uL T4PNK, 6uL dH₂O, mixing uniformly; heating at 37 deg.C for 30min, heating at 95 deg.C for 5min, gradient cooling to 25 deg.C, incubating at 25 deg.C for 5min, incubating at 4 deg.C for 5min, and storing at-20 deg.C for use.

Even more preferably: the specific process of the step (4) is as follows:

(b) adding 3mL of buffer E into the electrotransfer glass tube, and inserting the electrotransfer glass tube into a pipettor seat;

(d) and placing the cells after the electrotransformation into a 6-hole plate, and culturing for 72h to realize knockout.

The room temperature of the invention is 25 +/-5 ℃.

The invention has the following benefits:

1. the gene is edited by aiming at the primary pig cells, and the primary pig cells of large animals are relatively difficult to transfect, so that the gene is difficult to edit, and related researches are few.

2. The most effective sgRNA target sequence is screened by RGS-CR, and the accuracy and reliability of gene editing are improved.

Drawings

FIG. 1 is a fluorescent plot of the detection of px459-sgRNA + RGS-sgRNA;

FIG. 2 is a fluorescence diagram showing the electrotransfer efficiency of px458-EGFP under different electrotransfer conditions;

FIG. 3 shows site-directed mutagenesis of a target gene in porcine fetal fibroblasts obtained by sequencing.

Detailed Description

In order to facilitate understanding of the technical scheme of the invention, the following provides a further description of a method for realizing gene knockout in porcine fetal fibroblasts by using the CRISPR/Cas9 system in combination with specific experimental examples.

Experimental example 1: a method for realizing gene knockout in porcine fetal fibroblasts by using a CRISPR/Cas9 system comprises the following steps:

the invention provides a method for realizing gene knockout in porcine fetal fibroblasts by using a CRISPR/Cas9 system, which comprises the following steps of transfecting a px459v2.0-Cas9 plasmid containing a specific sgRNA targeting sequence to PEF by using a Neon electrotransformation system to obtain a PEF cell with gene knockout, wherein the method comprises the following steps:

determination of target sequence of target gene and construction of Cas9 plasmid

Among exon sequences of the target gene, the PAM sequence, i.e., N, is found₍₂₀₎NGG，N₍₂₀₎Namely the target sequence of the target gene. The target gene selected in the invention is pROSA26, different target sequences of sgRNA1 and sgRNA2 are designed according to the target gene, the target sequence of the sgRNA1 is 5'-AAAGTGTGCTGTGTATTTTG-3' (SEQ ID No.1), and the target sequence of the sgRNA2 is 5'-AGAGAAGAGGCTGTGCTCTG-3' (SEQ ID No. 2);

sending the sgRNA1 target sequence together with the cohesive end 5'-CACCGAAAGTGTGCTGTGTATTTTG-3' (SEQ ID No.3) of four bases of the restriction enzyme BbsI and the complementary sequence together with the cohesive end 5'-AAACCAAAATACACAGCACACTTTC-3' (SEQ ID No.4) of four bases of the restriction enzyme BbsI, the target sequence of the sgRNA2 together with the cohesive end 5'-CACCGAGAGAAGAGGCTGTGCTCTG-3' (SEQ ID No.5) of four bases of the restriction enzyme BbsI and the complementary sequence together with the cohesive end 5'-AAACCAGAGCACAGCCTCTTCTCTC-3' (SEQ ID No.6) of four bases of the restriction enzyme BbsI to Beijing Optimalaceae, Inc. for synthesis;

the synthesized target sequence and its complementary sequence were dissolved in water to 100uM, 1uL each was taken, and 1uL 10 × T4ligase buffer, 1uL T4PNK, 6uL dH were added₂O, mixing uniformly; heating at 37 deg.C for 30min, heating at 95 deg.C for 5min, gradient cooling to 25 deg.C, incubating at 25 deg.C for 5min, incubating at 4 deg.C for 5min to form DNA double strand, and storing at-20 deg.C;

the plasmid px459v2.0-cas9 (purchased from addrene) was digested with the restriction enzyme Bbs I (NEB) in the reaction system of 10uL of DNA plasmid, 5uL of Bbs I restriction enzyme, 10 XBuffer G10 uL and 175uL of deionized water, mixed and incubated at 37 ℃ for 3 h. After incubation, running 1.2% agarose Gel, separating the target fragment, about 9000bp, recovering the target DNA fragment with agarose Gel recovery Kit (AxyPrep DNA Gel Extraction Kit), ensuring the concentration at 50ng/uL, and storing at-20 ℃ for later use;

the target sequence and the complementary double-stranded DNA are cloned into a plasmid px459v2.0-cas9 cut by a restriction endonuclease BbsI (NEB), and the connecting system is as follows: the recovered plasmid backbone of px459v2.0-cas9 (100 ng), the target sequence and complementary DNA double strand (2 uL), 10 XT 4 DNA ligase buffer (1 uL), T4ligase (1 uL) and deionized water are added to make up to 10 uL. Ligation was carried out overnight at 16 ℃. The ligation product is introduced into a competent cell DH5a for transformation, amplification culture and extraction of plasmids after correct sequencing, the obtained plasmids are named as px459-sgRNA1 and px459-sgRNA2 respectively, and the concentration of the plasmids is adjusted to 1 ug/uL.

Construction of RGS-CR plasmid containing target point sequence and screening of different target sequence activities of target gene in 293T cell

1. Construction of RGS-CR plasmids containing target sequences

The RGS-CR reporter sequence of the sgRNA1 target sequence is 5 '-AAAGTGTGCTGTGTATTTTGAGG(PAM) -3' (SEQ ID No.7), and is named sgRNA 1-RGS-CR; the RGS-CR reporter sequence of the sgRNA2 target sequence is 5 '-AGAGAAGAGGCTGTGCTCTGGGG(PAM) -3' (SEQ ID No.8), and is named as sgRNA 2-RGS-CR; RGS-CR report vector sequences of sgRNA1 and sgRNA2 targets and sticky ends of four bases of restriction enzymes EcoRI and BamHI are sent to Beijing Strongobike Limited to synthesize; wherein the sgRNA1 target sequence together with the EcoRI sticky end sequence is 5' -AATTCAAAGTGTGCTGTGTATTTTGAGG(PAM) -3 ' (SEQ ID No.9) whose reverse complement together with the cohesive end sequence of the restriction enzyme BamH I is 5'-GATCCCTCAAAATACACAGCACACTTTG-3' (SEQ ID No. 10); the sgRNA2 point sequence together with the EcoRI cohesive end sequence was 5' -AATTCAGAGAAGAGGCTGTGCTCTGGGG(PAM)-3 ' (SEQ ID No.11) the complement of which together with the BamH i cohesive end sequence is 5'-GATCCCCCAGAGCACAGCCTCTTCTCTG-3' (SEQ ID No. 12);

RGS-CR reporter vector (double-fluorescent reporter plasmid disclosed in the prior art) was digested simultaneously with restriction enzymes EcoRI and BamHI, in the order of 20ug, EcoRI 10uL, BamHI 10uL, 10 XTango buffer 40uL of RGS-CR plasmid, and DEPC water 120 uL. Incubate at 37 ℃ for 3h and recover the plasmid backbone. The sgRNA1-RGS-CR and the sgRNA2-RGS-CR were ligated to the RGS-CR backbone, and the resulting vectors were designated RGS-sgRNA1 and RGS-sgRNA2, respectively.

2. Screening of activity of different target sequences of target gene in 293T cell

293T cells were transfected with the px459-sgRNA1 plasmid, px459-sgRNA2, RGS-CR, px459-sgRNA1+ RGS-sgRNA1, px459-sgRNA2+ RGS-sgRNA2, respectively. On the day before transfection, 293T cells were transferred to 5 60mm dishes on average and transfected when their confluence reached 90%. Taking 10 1.5mL centrifuge tubes, and numbering 1-10 respectively; adding 280uL opti-M into a No. 1-5 centrifuge tube, then adding 16.8uL lipo3000 transfection reagent, and uniformly mixing; adding 280uL opti-M, then 24uL P3000, then adding 12ug px459-sgRNA1 in a No.6 tube, adding 12ug px459-sgRNA2 in a No.7 tube, adding 12ug RGS-CR in a No.8 tube, adding 6ug px459-sgRNA1 and 6ug RGS-sgRNA1 in a No.9 tube, and adding 6ug px459-sgRNA2 and 6ug RGS-sgRNA2 in a No.10 tube; after mixing, the transfection reagent in tube 1 was added to tube 6, tube 2 was added to tube 7, tube 3 was added to tube 8, tube 4 was added to tube 9, and tube 5 was added to tube 10, and the mixture was mixed well to form a transfection mixture, which was left to stand at room temperature for 15min and then added dropwise to 293T cells. After 48h, fluorescence expression was observed (FIG. 1). Fluorescence showed that the green fluorescence expression of px459-sgRNA2+ RGS-sgRNA2 was significantly greater than that of px459-sgRNA1+ RGS-sgRNA1, and thus px459-sgRNA2 was subsequently selected for subsequent targeting experiments.

Thirdly, utilizing Neon^TMThe electrotransfer system carries out electrotransfer condition screening and the px459-sgRNA2 is electrotransferred to PEF to realize knockout

1、Neon^TMElectrotransfer px458-EGFP for electrotransfer condition screening

Taking 2 Pig Adult Fibroblast (PAF) with 35mm culture dish, digesting with Tryple, centrifuging at 1000rpm for 5min, and collecting cell precipitate; resuspending the cells in 1mL PBS, transferring the cells into a 1.5mL centrifuge tube, and centrifuging to remove DPBS; adding 36uL Buffer R into cell precipitates of 2 35mm culture dishes, adding 4uL px458-EGFP plasmids, and uniformly mixing; adding 1mL of non-resistant cell culture medium into a 12-well plate, and preheating an incubator at 37 ℃; beating the electric transfer liquid gun to 2 grades to completely loosen the screw, forcibly inserting the gun head into the liquid transfer device to completely clamp the metal strip in the gun head by the screw, and loosening a button of the liquid transfer device; pressing the gun to the first gear, and slowly sucking air to mix the liquid by 10 uL; putting the liquid transfer gun into a glass tube, hearing a click, adjusting voltage, pulse time and pulse frequency, starting according to start, taking out the liquid transfer gun after completing electric shock and completing complete, adding mixed liquid in the gun into a 12-hole plate, and immediately shaking up; to select the optimum conditions, the test was performed 3 sets of 1500V, 10ms, 3 times, 1600V, 10ms, 2 times, 1650V, 20ms, 1 time. After 48h incubation, the transfection efficiency was determined by observing green fluorescence, while the cell viability was determined by observing the cell status (FIG. 2). The results show that the cell transfection efficiency is high and the cell viability is good under the condition of 1500V, 10ms and 3 times of electrotransformation, so that the condition is selected for electrotransformation in subsequent experiments.

2. Knock-out by electrotransformation of px459-sgRNA2 to PEF

Digesting the PEF transferred to the third generation for 3min by Tryple, collecting the PEF into a 15mL centrifuge tube, and centrifuging the PEF for 5min at 1000 rpm; at the moment, 2mL of non-resistant culture medium is added into each hole of the 6-hole plate, and the 6-hole plate is placed into an incubator at 37 ℃ in advance for preheating; adding buffer E into the electric rotating glass tube, and inserting a pipettor seat; after PEF centrifugation, using DPBS to clean once, centrifuging for 5min, discarding DPBS, using 9uLBuffer R to resuspend cell sediment, adding 2ug px459-sgRNA2, mixing uniformly, performing electrotransformation for 3 times at 1500V for 10ms, adding the electrotransformed cells into a 6-well plate, and culturing for 72h to realize knockout;

detection of mutant PEF

The method mainly detects the knockout efficiency of pig fetal fibroblast ROSA26 mediated by CRISPR/Cas9 system, and comprises the following steps:

after the cells subjected to electric transformation are cultured for 72h, pancreatin is digested, the cells are centrifuged at 1000rpm for 5min, and the pancreatin is discarded; washing with PBS once, centrifuging at 1000rpm for 5min, discarding PBS, and collecting cell precipitate; extracting PEF genomic DNA using a genomic DNA extraction kit (tiangen DP 304);

amplifying a DNA fragment of about 500bp near a target point by utilizing a PCR upstream primer and a PCR downstream primer, wherein the PCR system comprises the following steps: 12.5uL of Q5 high-fidelity enzyme, 1.25uL of each of upstream and downstream primers (SEQ ID No.13 and SEQ ID No.14), 1uL of genomic DNA and 9uL of deionized water; the amplification procedure was: 30s at 98 deg.C, 35 × (5 s at 98 deg.C, 10s at 58 deg.C, 20s at 72 deg.C), 2min at 72 deg.C, and storing at 4 deg.C; the primers used were as follows:

sequence 13: ACTGTGTTGGCGGACTGG (SEQ ID No.13)

Sequence 14: AACGATATGCTCCCGACTTCC (SEQ ID No.14)

And connecting the amplified PCR product with a T vector, and sequencing. And (3) selecting 19 clones for sequencing, carrying out mutation, wherein the mutation rate is 15.79%, and the detection result of the PEF mutation is shown in figure 3.

The method for realizing gene knockout in porcine fetal fibroblasts by using the CRISPR/Cas9 system can realize efficient gene editing on porcine primary cells.

Sequence listing

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