长效CRISPR/Cas9基因编辑结局的动态追踪研究

doi:10.11843/j.issn.0366-6964.2023.10.018

摘要/Abstract

摘要： 旨在通过构建体外细胞模型研究在长效CRISPR系统作用下基因编辑的动态结局，为在体生殖系长效编辑提供研究支持。本研究以FANCF和VEGFA基因为例，通过慢病毒随机整合的方式构建了一套长效表达CRISPR/Cas9的细胞模型，并以该模型为对象研究长效CRISPR系统作用下，基因编辑效率和修复的动态结局。在FANCF和VEGFA基因外源靶点区域，随着Doxycycline诱导时间的增加，CRISPR/Cas9的编辑效率逐渐升高，并分别于第4天（FANCF）、第3天（VEGFA）编辑效率达到峰值；其次，对应上述靶区的内源靶点的达到峰值的时间点与外源靶点基本一致，但内源靶点的编辑效率显著高于外源靶点（P<0.000 1）；再次，对靶点编辑产物序列分析发现，在编辑的第4~7天，各靶点编辑产物的删除、插入及其它类型呈稳定状态，内、外源各位点编辑结局中删除、插入以及其它类型的占比趋势一致；最后，对FANCF靶点潜在的6个脱靶位点进行检测，均未发现任何脱靶现象。长效CRISPR/Cas9技术的编辑效率在第3或第4天达到峰值，各基因编辑结局类型占比趋势一致，且在FANCF位点上不会增加脱靶风险。

关键词: CRISPR/Cas9, 基因编辑, 动物育种

Abstract: The aim was to study the dynamic outcome of gene editing under the action of a long-acting CRISPR system by constructing an in vitro cellular model to provide research support for long-acting editing in germ line. A set of cellular model expressing CRISPR/Cas9 in a long-term manner was constructed by lentiviral random integration of FANCF and VEGFA genes, and this model was used to study the dynamic outcome of gene editing efficiency and repair under the action of the long-term CRISPR system. In the exogenous target region of FANCF and VEGFA genes, the editing efficiency of CRISPR/Cas9 gradually increased with the increase of Doxycycline treatment time, and respectively reached the highest on day 4 (FACNF) and day 3 (VEGFA); secondly, the peak time points of the endogenous target corresponding to the above target region was basically the same as that of the exogenous target, but the editing efficiency of the endogenous target was significantly higher than that of the exogenous target (P<0.000 1); thirdly, the analysis of the sequence of the target editing products revealed that the deletion, insertion and other types of the target editing products were in a stable state from day 4 to 7 of editing, and the trend of deletion, insertion and other types in the editing ending of each endogenous target and exogenous target were consistent; finally, the potential 6 off-target sites of the FANCF targets were detected, and no off-target phenomenon was found. The editing efficiency of the long-term CRISPR/Cas9 technology peaked at day 3 or 4, with a consistent proportion trend of gene editing outcome types across endogenous targets and exogenous targets, and no increased off-target risk at the FANCF locus.

Key words: CRISPR/Cas9, gene editing, animal breeding

中图分类号:

S811.3

张硕, 周雨潇, 吴海波, 索伦. 长效CRISPR/Cas9基因编辑结局的动态追踪研究[J]. 畜牧兽医学报, 2023, 54(10): 4196-4208.

ZHANG Shuo, ZHOU Yuxiao, WU Haibo, SUO Lun. Dynamics of Gene Editing Consequence Mediated by Long-term CRISPR/Cas9 System[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4196-4208.

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