基于小型化Cas蛋白的CRISPR/Gal4BD-Cas供体适配基因编辑系统研究

doi:10.16288/j.yczz.24-124

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Hereditas(Beijing) ›› 2024, Vol. 46 ›› Issue (9): 716-726.doi: 10.16288/j.yczz.24-124

• Research Article • Previous Articles Next Articles

CRISPR/Gal4BD-Cas donor adapting systems based on miniaturized Cas proteins for improved gene editing

Sen Yang(), Baoxia Ma, Hongrun Qian, Jieyu Cui, Xiaojun Zhang, Lida Li, Zehui Wei, Zhiying Zhang, Jiangang Wang(), Kun Xu()

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

Received:2024-06-28 Revised:2024-08-24 Online:2024-08-26 Published:2024-08-26
Contact: Jiangang Wang, Kun Xu E-mail:yangsen@nwafu.edu.cn;wangjiangang@126.com;xukunas@nwafu.edu.cn
Supported by:
Biological Breeding-Major Projects(2023ZD04074);Biological Breeding-Major Projects(2023ZD04051)

Abstract

Abstract:

Targeted precise point editing and knock-in can be achieved by homology-directed repair(HDR) based gene editing strategies in mammalian cells. However, the inefficiency of HDR strategies seriously restricts their application in precision medicine and molecular design breeding. In view of the problem that exogenous donor DNA cannot be efficiently recruited autonomously at double-stranded breaks(DSBs) when using HDR strategies for gene editing, the concept of donor adapting system(DAS) was proposed and the CRISPR/Cas9-Gal4BD DAS was developed previously. Due to the large size of SpCas9 protein, its fusion with the Gal4BD adaptor is inconvenient for protein expression, virus vector packaging and in vivo delivery. In this study, two novel CRISPR/Gal4BD-SlugCas9 and CRISPR/Gal4BD-AsCas12a DASs were further developed, using two miniaturized Cas proteins, namely SlugCas9-HF derived from Staphylococcus lugdunensis and AsCas12a derived from Acidaminococcus sp. Firstly, the SSA reporter assay was used to assess the targeting activity of different Cas-Gal4BD fusions, and the results showed that the fusion of Gal4BD with SlugCas9 and AsCas12a N-terminals had minimal distraction on their activities. Secondly, the HDR efficiency reporter assay was conducted for the functional verification of the two DASs and the corresponding donor patterns were optimized simultaneously. The results demonstrated that the fusion of the Gal4BD adaptor binding sequence at the 5′-end of intent dsDNA template (BS-dsDNA) was better for the CRISPR/Gal4BD-AsCas12a DAS, while for the CRISPR/Gal4BD-SlugCas9 DAS, the dsDNA-BS donor pattern was recommended. Finally, CRISPR/Gal4BD-SlugCas9 DAS was used to achieve gene editing efficiency of 24%, 37% and 31% respectively for EMX1, NUDT5 and AAVS1 gene loci in HEK293T cells, which was significantly increased compared with the controls. In conclusion, this study provides a reference for the subsequent optimization of the donor adapting systems, and expands the gene editing technical toolbox for the researches on animal molecular design breeding.

Key words: gene editing, homology-directed repair, donor adapting, miniaturized Cas proteins, adaptor

Sen Yang, Baoxia Ma, Hongrun Qian, Jieyu Cui, Xiaojun Zhang, Lida Li, Zehui Wei, Zhiying Zhang, Jiangang Wang, Kun Xu. CRISPR/Gal4BD-Cas donor adapting systems based on miniaturized Cas proteins for improved gene editing[J]. Hereditas(Beijing), 2024, 46(9): 716-726.

Figures/Tables 6

Table 1

Table 2

Fig. 1

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Fig. 4

References 37

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蛋白名称	蛋白来源	类型	蛋白长度	PAM序列	gRNA类型	参考文献
SaCas9	Staphylococcus aureus	II-A	1053 aa	-NNGRRT	sgRNA	[18]
SchCas9	Staphylococcus chromogenes	II-A	1054 aa	-NNGR	tracrRNA	[19]
SlugCas9	Staphylococcus lugdunensis	II-A	1054 aa	-NNGG	sgRNA	[20]
CjCas9	Campylobacter jejuni	II-C	984 aa	-NNNNRYAC	sgRNA	[21]
NmeCas9	N. meningitidis strain 8013	II-C	1081 aa	-NNNNGATT	sgRNA	[22]
AsCas12a	Acidaminococcus	V-A	1307 aa	TTTV-	crRNA	[16]
LbCas12a	Lachnospiraceae bacterium	V-A	1228 aa	TTTV-	crRNA	[17]
Cas12c	−	V-C	1209~1330 aa	5′TG/5′TN	tracrRNA	[27]
Cas12j(Casφ)	Biggiephage	V-J	700~800 aa	TTN-	crRNA	[23]
AsCas12f1	Acidibacillus sulfuroxidans	V-F	422 aa	NTTR-	sgRNA	[24]
SpCas12f1	Syntrophomonas palmitatica	V-F	497 aa	TTC-	sgRNA	[25]
CasMINI	Engineering from Un1Cas12f1	V-F	529 aa	TTTR-	sgRNA	[26]
Cas12h	−	V-H	870~924 aa	5′RTR	crRNA	[27]
Cas12i	−	V-I	1033~1093 aa	5′TTN	crRNA	[27]
Cas12g	−	V-G	720~830 aa	无	crRNA、tracrRNA	[27]

引物名称	引物序列(5′→3′)
EMX1-F	TTCTCCTGACTGTTCCTTGT
EMX1-R	GGCACGTTGCTCTTTCTTGGGC
NUDT5-F	CTTTTACCCTAAAACTGGAAAATCAGTCTTGCTA
NUDT5-R	GCAGAGCAAACCGTCTTCTCCATTCA
AAVS1-F	ATCGAATTCAGCCGGTCCTGGACTTTGTC
AAVS1-R	GGCTGGCTACTGGCCTTATC

CRISPR/Gal4BD-Cas donor adapting systems based on miniaturized Cas proteins for improved gene editing

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