不同CRISPR/Cas9供体适配基因编辑系统的比较及优化研究

doi:10.16288/j.yczz.23-273

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Hereditas(Beijing) ›› 2024, Vol. 46 ›› Issue (6): 466-477.doi: 10.16288/j.yczz.23-273

• Research Article • Previous Articles Next Articles

Comparison and optimization of different CRISPR/Cas9 donor-adapting systems for gene editing

Ma Baoxia¹(), Yang Sen¹, Lyu Ming¹, Wang Yuren¹, Chang Liye¹, Han Yifan¹, Wang Jiangang¹, Guo Yang²(), Xu Kun¹()

1. College of Animal Science and Technology, Northwest A&F University, Yangling 71200, China
2. Xinjiang Uygur Autonomous Region Animal Husbandry General Station, Urumchi 830002, China

Received:2024-01-07 Revised:2024-03-22 Online:2024-06-20 Published:2024-03-27
Supported by:
Scientific and Technological Innovation 2030?Major Project of Agricultural Biological Breeding(2023ZD04074);Scientific and Technological Innovation 2030?Major Project of Agricultural Biological Breeding(2023ZD04051)

Abstract

Abstract:

Gene knock-in in mammalian cells usually uses homology-directed repair (HDR) mechanism to integrate exogenous DNA template into the target genome site. However, HDR efficiency is often low, and the co-localization of exogenous DNA template and target genome site is one of the key limiting factors. To improve the efficiency of HDR mediated by CRISPR/Cas9 system, our team and previous studies fused different adaptor proteins with SpCas9 protein and expressed them. By using their characteristics of binding to specific DNA sequences, many different CRISPR/SpCas9 donor adapter gene editing systems were constructed. In this study, we used them to knock-in eGFP gene at the 3′-end of the terminal exon of GAPDH and ACTB genes in HEK293T cells to facilitate a comparison and optimization of these systems. We utilized an optimized donor DNA template design method, validated the knock-in accuracy via PCR and Sanger sequencing, and assessed the efficiency using flow cytometry. The results showed that the fusion of yGal4BD, hGal4BD, hLacI, hTHAP11 as well as N57 and other adaptor proteins with the C-terminus of SpCas9 protein had no significant effect on its activity. At the GAPDH site, the donor adapter systems of SpCas9 fused with yGal4BD, hGal4BD, hLacI and hTHAP11 significantly improved the knock-in efficiency. At the ACTB site, SpCas9 fused with yGal4BD and hGal4BD significantly improved the knock-in efficiency. Furthermore, increasing the number of BS in the donor DNA template was beneficial to enhance the knock-in efficiency mediated by SpCas9-hTHAP11 system. In conclusion, this study compares and optimizes multiple CRISPR/Cas9 donor adapter gene editing systems, providing valuable insights for future gene editing applications.

Key words: gene editing, gene knock-in, CRISPR/Cas9, donor adapting, homology-directed repair

Ma Baoxia, Yang Sen, Lyu Ming, Wang Yuren, Chang Liye, Han Yifan, Wang Jiangang, Guo Yang, Xu Kun. Comparison and optimization of different CRISPR/Cas9 donor-adapting systems for gene editing[J]. Hereditas(Beijing), 2024, 46(6): 466-477.

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基因名称	靶序列	PAM
GAPDH	CCTCCAAGGAGTAAGACCCC	TGG
	CATGGCCCACATGGCCTCCA	AGG
	TCTAGGTATGACAACGAATT	TGG
ACTB	CGTCCACCGCAAATGCTTCT	AGG
	AAGCAGGAGTATGACGAGTC	CGG
	GATGGAGCCGCCGATCCACA	CGG

名称	结合序列(5′→3′)
yGal4BD	TCCGGAGGACTGTCCTCCGG
hGal4BD	TCCGGAGGACTGTCCTCCGG
hTHAP11	AGGACTACATTTCCCAGCA
hLacI	GGAATTGTTATCCGCTCACAATTCC
N57	TACAGTTGAAGTCGGAAGTTTACATACACTT AAGTTGGAGTCATTAAAACTCGTTTTTCAAC TACTCCACAAATTTCTTGTTAACAAACAATA GTTTTGGCAAGTCAGTTAGGACATCTACTTT GTGCATGACACAAGTCATTTTTCCAACAATT GTTTACAGACAGATTATTTCACTTATAATTC ACTGTATCACAATTCCAGTGGGTCAGAAGTT TACATACACTAAGTTGACTGTGCCTTTAAAC AGCTTGGAAAATTCCAGAAAATGATGTCATG GCTTTAGAAGCTT

名称	引物序列(5′→3′)
Detection-G-F1	CAGGACCCGGGTTCATAACT
Detection-G-R1	CGCTGAACTTGTGGCCGTTTA
Detection-A-F1	CACACTTAGCCGTGTTCTTTGC
Detection-A-R1	TTGCCGTAGGTGGCATCGCCC

Comparison and optimization of different CRISPR/Cas9 donor-adapting systems for gene editing

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