提高CRISPR/Cas9介导的动物基因组精确插入效率 研究进展

doi:10.16288/j.yczz.20-056

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Hereditas(Beijing) ›› 2020, Vol. 42 ›› Issue (7): 641-656.doi: 10.16288/j.yczz.20-056

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Recent developments in enhancing the efficiency of CRISPR/Cas9- mediated knock-in in animals

Guoling Li¹, Shanxin Yang¹, Zhenfang Wu^1,², Xianwei Zhang²()

^1. National Engineering Research Center for Swine Breeding Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China
^2. Wens Foodstuff Co., Ltd., Xinxing 527439, China

Received:2020-03-04 Revised:2020-04-24 Online:2020-07-20 Published:2020-06-01
Contact: Zhang Xianwei E-mail:zxianw@163.com
Supported by:
Supported by the National Transgenic Major Projects No(2016ZX08006002)

Abstract

Abstract:

Gene-editing technology can artificially modify genetic material of targeted loci by precise insertion, deletion, or replacement in the genomic DNA. In recent years, with the developments of zinc-finger endonuclease (ZFN), transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats/CRISPR- associated protein 9 (CRISPR/Cas9) technologies, such precise modifications of the animal genomes have become possible. Although gene-editing tools, such as CRISPR/Cas9, can efficiently generate double-strand breaks (DSBs) in mammalian cells, the homology-directed repair (HDR) mediated knock-in (KI) efficiency is extremely low. In this review, we briefly describe the current development of gene-editing tools and summarize the recent strategies to enhance the CRISPR/Cas9- mediated KI efficiency, which will provide a reference for the generation of human disease models, research on gene therapy and livestock genetic improvement.

Key words: gene editing, CRISPR/Cas9, knock in, homology directed repair, non-homologous end joining

Guoling Li, Shanxin Yang, Zhenfang Wu, Xianwei Zhang. Recent developments in enhancing the efficiency of CRISPR/Cas9- mediated knock-in in animals[J]. Hereditas(Beijing), 2020, 42(7): 641-656.

Figures/Tables 1

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小分子化合物	细胞类型	浓度(µmol/L)	供体模板	KI效率	参考文献
Scr7 (NHEJ抑制剂)	家兔胚胎	40	dsDNA	None	[2]
	A549、MelJuSo、小鼠受精卵	0.01、1、1	dsDNA、ssODN	2~19倍	[7]
	HEK293	1	dsDNA	5倍	[65]
	hiPSCs	1	dsDNA	None	[66]
	HEK293T	10	dsDNA	None	[71]
	PFFs	100	dsDNA	1.9倍	[98]
	HEK293A	1	dsDNA	None	[102]
	HEK293T	1	dsDNA	1.8倍	[104]
	CHO	0.1~20	dsDNA	None	[108]
	PFFs	5	dsDNA	None	[115]
	小鼠受精卵	50	ssODN	9.7倍	[120]
	斑马鱼胚胎	20	dsDNA、ssODN	2.5~3.7倍	[121]
	PFFs	1	ssODN	None	[122]
	hiPSCs	1	ssODN	None	[123]
L755507 (机理不清楚)	hiPSCs	5	dsDNA	None	[66]
	PFFs	40	dsDNA	1.9倍	[98]
	mESCs、HUVEC、 K562、Hela、FCRL-2097、NSCs、hiPSCs	5	dsDNA、ssODN	1.3~8.9倍	[101]
	HEK293A	5	dsDNA	None	[102]
	hiPSCs	5	ssODN	None	[114]
	hiPSCs	5	ssODN	1.8倍	[124]
	hiPSCs	NA	ssODN	None	[125]
	斑马鱼胚胎	2	ssODN	None	[126]
RS-1 (HDR激活剂)	家兔胚胎	7.5	dsDNA	2~5倍	[2]
	hiPSCs	10	dsDNA	None	[66]
	HEK293A、U2OS	20	dsDNA	3~6倍	[102]
	斑马鱼胚胎	20	dsDNA	1.6倍	[121]
	hiPSCs	1	ssODN	None	[123]
	斑马鱼胚胎	100	ssODN	None	[126]
Nocodazole (细胞周期阻滞)	hiPSCs	0.10	dsDNA	1.5倍	[66]
	HEK293T、HFF、hESCs	0.20	ssODN	提高至38%	[99]
	hiPSCs	1	dsDNA	3.8~5.8倍	[107]
	HEK293T	0.10	dsDNA、ssODN	None	[113]
	PFFs	1	dsDNA	2.8倍	[115]

Recent developments in enhancing the efficiency of CRISPR/Cas9- mediated knock-in in animals

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