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Method for improving eukaryotic gene editing efficiency and application thereof

A technology of gene editing and eukaryotes, applied in biochemical equipment and methods, microorganisms, genetic engineering, etc.

Pending Publication Date: 2022-05-17
SHANDONG UNIV
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This strategy increased the activity of nearly all gRNAs tested located in open chromatin, whereas gRNAs located in heterochromatin regions had no promoting effect for one-third of the tested gRNAs

Method used

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  • Method for improving eukaryotic gene editing efficiency and application thereof
  • Method for improving eukaryotic gene editing efficiency and application thereof
  • Method for improving eukaryotic gene editing efficiency and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0072] 1.1 Genome editing efficiency is related to chromatin opening state

[0073] The rs339331 locus is T / C heterozygous in two prostate cancer cell lines, VCaP and 22Rv1, and previous studies revealed that these two alleles have different chromatin opening states. Directly compare the genome editing efficiency of two different alleles near the rs339331 site ( figure 1 a), allowing the present invention to strip the potential impact of sgRNA target sequence differences when studying the correlation between genome editing efficiency and chromatin open state. Sanger sequencing results of FAIRE DNA in VCaP cells ( figure 1 b) Sanger sequencing results of FAIRE DNA in and 22Rv1 cells both showed T allele enrichment. FAIRE qPCR analysis of these two cell lines using allele-specific primers confirmed that the T allele has a more open chromatin state than the C allele ( figure 1 c). In VCaP ( figure 1 d) The indel frequency of the T allele is significantly higher than that of ...

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Abstract

The invention provides a method for improving eukaryote gene editing efficiency and application thereof, and belongs to the technical field of biology. According to the invention, how the chromatin accessibility affects the genome editing efficiency of CRISPR / Cas9 in mammalian cells is systematically studied, and the genome editing efficiency of Cas9 is improved by fusing Cas9 with a transcriptional activation domain. Moreover, the accessibility of chromatin is increased by using YF-2, and the YF-2 is a high-selectivity HAT activator and has the activity aiming at CBP, PCAF and GCN5. Researches prove that the genome editing activity of Cas9-VP64 can be further improved under the condition that the relative off-target effect is not increased by using the HAT activator. The invention provides a new strategy for improving the CRISPR / Cas9 genome editing activity, and makes it possible to select a wider range of gRNA targets in eukaryotes.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for improving eukaryotic gene editing efficiency and its application. Background technique [0002] The information disclosed in the Background of the Invention is only intended to increase the understanding of the general background of the invention, and is not necessarily to be taken as an acknowledgment or any form of suggestion that the information constitutes the prior art that is already known to those skilled in the art. [0003] The RNA-guided CRISPR / Cas9 nuclease system has become the first choice for genome editing research and has broad prospects in biotechnology and clinical applications. However, when CRISPR / Cas9 is used in eukaryotes, it exhibits widely varying editing efficiencies on different targets and even on the same target in different cell types. As researchers look for more efficient gRNA targets for all uses, uncovering the factors involv...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/113C12N5/10C07K19/00
CPCC12N15/85C12N9/22C12N15/113C12N5/0686C12N2501/065C12N2510/00C12N2310/20C12N2800/107C07K2319/71
Inventor 黄启来刘俊豪李博
Owner SHANDONG UNIV
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