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CRISPR-SaCas9 gene editing system and application thereof

A technology for gene editing and encoding genes, applied in gene therapy, genetic engineering, DNA/RNA fragments, etc. It can solve the problem of low gene editing efficiency, and achieve the effect of improving gene knock-in efficiency, low off-target efficiency and broad application prospects.

Active Publication Date: 2021-10-26
天津协和生物科技开发有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, compared with the commonly used SpCas9, the gene editing efficiency of SaCas9 is lower

Method used

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  • CRISPR-SaCas9 gene editing system and application thereof
  • CRISPR-SaCas9 gene editing system and application thereof
  • CRISPR-SaCas9 gene editing system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 vector construction

[0042] This example is used to construct each vector of the gene editing system described in the present invention.

[0043] (1) Construction of different nuclear localization signal SaCas9 vectors

[0044] Different nuclear localization signal sequences were obtained by PCR amplification, and then EF1 promoter, different nuclear localization signal sequences (SEQ ID NO.8-14), SaCas9 (encoded by SEQ ID NO. The nucleotide sequence of .15), Wpre and PolyA were spliced ​​and cloned into the pEF1-SaCas9 plasmid; wherein, SaCas9 and the nuclear localization signal were connected by connecting sequence 1 (SEQ ID NO.16); digested with endonuclease and Sanger sequencing identified SaCas9 vectors with different nuclear localization signals.

[0045] (2) Construction of sgRNA vectors with different backbone structures and different target sequence lengths

[0046] Use the CHOPCHOP website (https: / / chopchop.rc.fas.harvard.edu / ) to design sgRNA...

Embodiment 2

[0051] Example 2 Gene editing method of human induced pluripotent stem cells at different sites

[0052] This example provides gene editing methods of human induced pluripotent stem cells at different sites.

[0053] (1) Electroporation of human induced pluripotent stem cells

[0054] Human induced pluripotent stem cells using mTeSR TM 1 culture medium. During recovery and passage, the culture plate was first coated with Matrigel. The specific steps are as follows: Before electroporation, iPSC cells were digested with Accutase to obtain a single cell suspension; 0.8-1.5×10 6 The cells were electroporated, and the amount of gene editing plasmids was as follows: 1 μg of Cas9, 0.5 μg of sgRNA (cutting the genome), 0.5 μg of sgDocut (cutting of pDonor), 1 μg of pDonor and 0.5 μg of BCL-XL plasmid; Human Stem Cell was used Kit 2 (Lonza) was used for electroporation, and the electroporation procedure was B-016.

[0055] (2) Gene editing of human induced pluripotent stem cells ...

Embodiment 3

[0063] Example 3 SaCas9 carrying nuclear localization signal improves gene editing efficiency

[0064] This example is used to compare the effects of different nuclear localization signals on the efficiency of SaCas9 gene editing. SaCas9 plasmids with different nuclear localization signals, including: Group 1: SaCas9 (No NLS) (1 μg), Group 2: SaCas9-NPM-NLS (1 μg), Group 3: SaCas9-BPNLS (1 μg), Group 4: BPNLS-SaCas9-BPNLS (1 μg) and Group 5: HMGA2-SaCas9-BPNLS (1 μg) (eg image 3 shown), co-electroporated iPSCs with CRISPR components such as pU6-sgEEF1A1 (0.5 μg), pD-EEF1A1-E2A-mNeonGreen-sg (1 μg), pU6-sgDocut (0.5 μg) and pEF1-BCL-XL (0.5 μg) , The gene knock-in efficiency was analyzed by FACS on the 3rd day after electroporation.

[0065] Such as Figure 4 and Figure 5 As shown, the HDR efficiency at EEF1A1 increased from 17% of the control SaCas9 (No NLS) to 30.2% (SaCas9-NPM-NLS), 29.9% (SaCas9-BPNLS), 25.8% (BPNLS-SaCas9-BPNLS) and 41 %(HMGA2-SaCas9-BPNLS). These ...

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Abstract

The invention provides a CRISPR-SaCas9 gene editing system and application thereof. The gene editing system comprises a SaCas9 vector, an sgRNA vector and a homologous recombination template pDonor vector. According to the gene editing system, the gene knock-in efficiency of mNeonGreen at an EEF1A1 locus and a GAPDH locus in a human induced pluripotent stem cell is improved by selecting appropriate methods such as a nuclear localization signal of the appropriate SaCas9 vector, a skeleton structure of the sgRNA vector and the target sequence length of the sgRNA vector. Meanwhile, the invention further systematically compares the targeting efficiency and off-target efficiency of SpCas9 and SaCas9 with different sgRNA lengths aiming at a same locus , and finally, it is learned that the CRISPR-SaCas9 gene editing system provided by the invention is a novel gene editing carrier system with high targeting efficiency and low off-target efficiency.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a gene editing system and its application, in particular to a CRISPR-SaCas9 gene editing system and its application. Background technique [0002] CRISPR-Cas9 is currently the most widely used gene editing tool. The currently commonly used CRISPR-Cas9 gene editing tool is mainly composed of two parts, namely the Cas9 protein responsible for cutting double-stranded DNA and the sgRNA responsible for recognizing the target DNA region through complementary base pairing. [0003] The working principle of the CRISPR-Cas9 gene editing system is as follows: [0004] The Cas9 protein first forms an RNP complex with the sgRNA, and the Cas9-sgRNA complex looks for the PAM sequence (Protospacer Adjacent Motif, protospacer Adjacent Motif) on the genome, and recognizes and temporarily stays at the PAM sequence. Once the target sequence on the sgRNA can be completely complemen...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/90C12N15/62C12N5/10A61K48/00A61K38/46
CPCC12N15/85C12N15/907C12N9/22C12N5/0696A61K48/005A61K48/0008A61K38/465C12N2800/107C12N2830/48C12N2830/50C07K2319/09Y02A50/30
Inventor 张孝兵程涛张健萍杨智学傅雅文
Owner 天津协和生物科技开发有限公司