一种新型提高HDR效率的CRISPR/Cas9-Gal4BD供体适配基因编辑系统

doi:10.16288/j.yczz.22-118

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Hereditas(Beijing) ›› 2022, Vol. 44 ›› Issue (8): 708-719.doi: 10.16288/j.yczz.22-118

• Research Article • Previous Articles Next Articles

A CRISPR/Cas9-Gal4BD donor adapting system for enhancing homology-directed repair

Xiaojun Zhang(), Kun Xu(), Juncen Shen, Lu Mu, Hongrun Qian, Jieyu Cui, Baoxia Ma, Zhilong Chen, Zhiying Zhang, Zehui Wei()

College of Animal Science and Technology, Northwest A&F University, Yangling 71200, China

Received:2022-04-05 Revised:2022-05-11 Online:2022-08-20 Published:2022-05-23
Contact: Xu Kun,Wei Zehui E-mail:mshn15@163.com;weizehui7848@163.com;xukunas@nwafu.edu.cn
Supported by:
the National Natural Science Foundation of China(32172736);the Shaanxi Key R&D Program(2021NY-027)

Abstract

Abstract:

The fast-rising CRISPR-derived gene editing technologies has been widely used in the fields of life science and biomedicine, as well as plant and animal breeding. However, the efficiency of homology-directed repair (HDR), an important strategy for gene knock-in and base editing, remains to be improved. In this study, we came up with the term Donor Adapting System (DAS) to summarize those CRISPR/Cas9 systems modified with adaptor for driving aptamer-fused donor DNA. A set of CRISPR/Cas9-Gal4BD DAS was designed in our study. In this system, Gal4 DNA binding domain (Gal4BD) is used as adaptor to fuse with Cas9 protein, and Gal4 binding sequence (Gal4BS) is used as aptamer to bind to the double-stranded DNA (dsDNA) donor, in order to improve the HDR efficiency. Preliminary results from the HEK293T-HDR.GFP reporter cell line show that the HDR editing efficiency could be improved up to 2-4 times when donor homologous arms under certain length (100-60 bp). Further optimization results showed that the choice of fusion port and fusion linker would affect the expression and activity of Cas9, while the Cas9-Gal4BD fusion with a GGS5 linker was the prior choice. In addition, the HDR efficiency was likely dependent on the aptamer-dsDNA donor design, and single Gal4BD binding sequence (BS) addition to the 5′-end of intent dsDNA template was suggested. Finally, we achieved enhanced HDR editing on the endogenous AAVS1 and EMX1 sites by using the CRISPR/Gal4BD-Cas9 DAS, which we believe can be applied to facilitate animal molecular design breeding in the future.

Key words: CRISPR/Cas9, gene editing, donor DNA, donor adapting, homology-directed repai

Xiaojun Zhang, Kun Xu, Juncen Shen, Lu Mu, Hongrun Qian, Jieyu Cui, Baoxia Ma, Zhilong Chen, Zhiying Zhang, Zehui Wei. A CRISPR/Cas9-Gal4BD donor adapting system for enhancing homology-directed repair[J]. Hereditas(Beijing), 2022, 44(8): 708-719.

Figures/Tables 6

Table 1

Fig. 1

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Fig. 4

Table 2

References 51

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分类	具体策略	检测所用细胞类型	不同类型供体提升效果		代表性参考文献
分类	具体策略	检测所用细胞类型	dsDNA	ssDNA	代表性参考文献
抑制NHEJ 通路	抑制DNA连接酶4	HEK293/K562/MEF/DC2.4/MelJuSo/HCT-116	2.4~19倍	~3倍	[5,17,18]
	抑制蛋白激酶催化亚基	HEK293/iPSC	2~5倍	1.6~3.3倍	[5,19,20]
	抑制Ku蛋白	HEK293/PFF	2~3倍	~2.4倍	[21,22]
	抑制53BP1	HEK293/Hela/K562/U2OS/LCL B/iPSC	1.4~3倍	1.3~3.3倍	[23,24]
增强HDR 通路	共表达CtIP转录因子	HEK293/iPSC	1.5~14.9倍	—	[25]
	共表达外切核酸酶1	K562/A549/H1299	2~2.5倍	—	[26]
	共表达Rad51、Rad52	HEK293/PK15/iPSC	2~6倍	1.4~2.4倍	[24,27]
优化供体形式	ssDNA供体优化	HEK293/U2OS/T cells	—	~3倍	[28,29]
	环状dsDNA供体优化	HEK293/HeLa/MCF10A/Embryo	1.1~18倍	—	[30,31]
	线性dsDNA供体优化	HEK293/iPSC	2~10倍	1.4~10倍	[32,33]
	供体与DBS共定位	HEK293	3~6倍	1.6~18倍	[11,12,34]
控制打靶时效	调节细胞周期	HEK293/iPSC/T cells	1.7~6倍	—	[35-37]
控制打靶时效	Cas9定时生效	HEK293/Two-cell embryo	1.87~10倍	1~5倍	[38,39]
其他机制	小分子化合物	HEK293/PFF/iPSC	2~3倍	2~10倍	[40]
其他机制	染色质状态	PFF/hES/iPSC/Embryo	~2倍	2-3倍	[41,42]

系统简称	融合端	Linker	适配配体	配体大小	适配受体	受体大小	供体形式	参考文献
Cas9-SNAP/ ssDNA	Cas9 C-terminus	Undeclared	SNAP-tag	181 aa	O6-BG	~241 (MW)	ssDNA	[9]
Cas9-Avidin/ dsDNA	Cas9	SGSETPGTSESATPES (16 aa)	Monomeric Streptavidin	114 aa	Biotin	~244 (MW)	dsDNA	[10]
Cas9-Avidin/ ssDNA	Cas9 C-terminus	SGSETPGTSESATPES (16 aa)	Avidin	152 aa	Biotin	~244 (MW)	ssDNA	[11]
sgRNA-S1m/ ssDNA	sgRNA	S1m*	Recombinant Streptavidin	159 aa	Biotin	~244 (MW)	ssDNA	[12]
Cas9-PVC/ ssDNA	Cas9 C-terminus	H4-2	Porcine Circovirus 2 (PCV) Rep	109 aa	PCV.BS	13 nt	ssDNA	[13]
Cas9-VirD2/ ssDNA (Plant editing)	Cas9	Undeclared	VirD2	455 aa	T-DNA.BS	25 nt	ssDNA	[14]
Cas9-THAP11/ dsDNA	Cas9 C-terminus	SGSETPGTSESATPES (16 aa)	THAP11	105 aa	THAP11.BS	19 bp	dsDNA	[15]
Cas9-N57/ dsDNA	Cas9 C-terminus	GGGGGSGGGGSGGGGSGGGGSLDPGGGGSG (30 aa)	N57	57 aa	N57.BS	292 bp	dsDNA	[16]
Cas9-Gal4BD/ dsDNA	Cas9 C-terminus	5×GGS (15 aa)	Gal4BD	146 aa	Gal4BD.BS	17 bp	dsDNA	This study

A CRISPR/Cas9-Gal4BD donor adapting system for enhancing homology-directed repair

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