Safe and efficient CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) gene editing technology

A gene editing and editing technology, applied in the field of gene editing, can solve problems affecting the process of clinical transformation and application, and achieve the effects of strong targeting, simple operation, and high cell transfection efficiency

Inactive Publication Date: 2018-11-13
SHENZHEN CHANGENE MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, as a gene editing technology, off-target effects are an inevitable shortcoming, which affects the process of its clinical translation application

Method used

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  • Safe and efficient CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) gene editing technology
  • Safe and efficient CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) gene editing technology
  • Safe and efficient CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) gene editing technology

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Experimental program
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Effect test

Embodiment 1

[0023] Hereinafter, the gene editing of HEK293T cells based on the hemophilia IX coagulation factor (F9) gene mutation site is described in detail as a specific gene editing scheme.

[0024] 1. For the F9 gene, design and synthesize 5 sgRNAs, whose sequences are shown in the table below.

[0025]

[0026] Among them, a total of 10 DNA sequences of sgRNA numbers 1-5 can be found in the sequence numbered 2 to 11 in the biological sequence table in the order of the above table, and their names correspond to the names in the above table.

[0027] Each pair of sgRNAs includes two sequences, one of which is the sense strand DNA (S) and the other is the antisense strand DNA (AS). The sequence underlined and in italics is the DNA sequence complementary to the F9 gene.

[0028] 2. First prepare the suspension of sgRNA DNA sense strand and antisense strand so that the final concentration is 100 μM, and anneal according to the following reaction system:

[0029] components ...

Embodiment 2

[0058] Analyze the off-target efficiency of psgRNA-F9-SpCas9-SZ.

[0059] We compared the F9-sgRNA-4 targeting sequence with other sequences in the human genome, and selected the sequence closest to the F9-sgRNA-4 targeting sequence (see Figure 6 sequence shown), specific primers were designed for these sequences, PCR amplification was carried out with genomic DNA as a template, and then the amplified product was sequenced, and the sequencing results (see Figure 7 ) showed that our psgRNA-F9-SpCas9-SZ had no cleavage at these sites.

Embodiment 3

[0061] The target gene was recombined into the F9 gene using psgRNA-F9-SpCas9-SZ and AAV targeting vector DNA, and the recombination efficiency was analyzed.

[0062] The pAAV-sgRNA-F9-EF1mini-SpCas9-SZ adeno-associated virus vector and the vector (pAAV-F9-Donor vector) containing the donor gene sequence for repairing the F9 gene were constructed and packaged to infect HEK293T cells. Fluorescence pictures of recombinant cells expressing EGFP are shown in Figure 8 .

[0063] The EGFP-positive cells were sorted by flow cytometry, the genomic DNA of the cells was extracted, and then the sequences between the homologous arms were amplified with specific primers, and the PCR products were sent for sequencing. For sequencing results, see Figure 9 . The results showed that the F9 gene was recombined successfully.

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Abstract

The invention aims at the technical defects of off-target effects of an existing CRISPR / Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR associated protein 9) gene editing technology to carry out site-directed mutation of an amino acid site on a site combined with a DNA (Deoxyribonucleic Acid) sequence of Cas9 protein to obtain the Cas9 protein with low off-target efficiency,so as to realize a gene editing unction with higher targeting performance and higher efficiency. Meanwhile, the invention further provides a method for carrying out HEK293T cell gene editing by adopting a gene editing method.

Description

technical field [0001] The invention relates to the technical field of gene editing, in particular to a safe and efficient CRISPR / Cas9 gene editing technology Background technique [0002] Clustered regulatory interspaced short palindromic repeat / CRISPR-associated genes (CRISPR / Cas) system is an acquired immune defense system evolved during the long-term resistance of bacteria to phages. Exist in bacteria and archaea. [0003] CRISPR is a unique type of DNA tandem repeat sequence. The CRISPR locus consists of a leader region (Leader), multiple short and highly conserved repeat sequence regions (Repeat) and multiple spacer sequence regions (Spacer). Cas genes are generally located near the CRISPR locus. According to the different sequences of the core elements of the Cas gene, the CRISPR / Cas immune system is divided into three types: type I, type II, and type III. Type I and Type III CRISPR / Cas immune systems require multiple Cas proteins to form a complex to cut DNA doubl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/10C12N15/113
CPCC12N9/22C12N15/102C12N15/113C12N2310/10
Inventor 姜舒纪惜銮张芸郭明
Owner SHENZHEN CHANGENE MEDICAL TECH CO LTD
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