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

Abstract

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

CLC Number:

S811.3

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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Dynamics of Gene Editing Consequence Mediated by Long-term CRISPR/Cas9 System

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