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

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

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

杨森(), 马宝霞, 钱泓润, 崔婕妤, 张潇筠, 李利达, 魏泽辉, 张智英, 王建刚(), 徐坤()

西北农林科技大学动物科技学院，杨凌 712100

收稿日期:2024-06-28 修回日期:2024-08-24 出版日期:2024-08-26 发布日期:2024-08-26
通讯作者: 王建刚，博士，讲师，研究方向：动物分子设计育种。E-mail: wangjiangang@126.com；
徐坤，博士，副教授，研究方向：动物基因编辑技术。E-mail: xukunas@nwafu.edu.cn
作者简介:杨森，硕士研究生，专业方向：动物生物技术。E-mail: yangsen@nwafu.edu.cn
基金资助:
农业农村部农业生物育种重大专项(2023ZD04074);农业农村部农业生物育种重大专项(2023ZD04051)

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 Published:2024-08-26 Online:2024-08-26
Supported by:
Biological Breeding-Major Projects(2023ZD04074);Biological Breeding-Major Projects(2023ZD04051)

摘要/Abstract

摘要：

在哺乳动物细胞中，利用同源引导修复(homology-directed repair，HDR)机制的基因编辑策略能够实现精准的点编辑和敲入，但是HDR的低效性严重制约了该策略在精准医疗和分子设计育种中的应用。鉴于HDR机制所需的供体DNA模板不能自主募集到基因组双链断裂(double-stranded break，DSB)处，本课题组提出了供体适配系统(donor adapting system，DAS)的概念并开发出CRISPR/SpCas9-Gal4BD供体适配基因编辑系统。由于SpCas9蛋白分子较大，与Gal4BD适配器结合不利于表达、病毒载体包装及活体递送等过程，因此本研究进一步采用两种小型化Cas蛋白——路邓葡萄球菌(Staphylococcus lugdunensis)源SlugCas9变体SlugCas9-HF与氨基酸球菌(Acidaminococcus sp.)源AsCas12a，开发了新型的CRISPR/Gal4BD-SlugCas9和CRISPR/Gal4BD-AsCas12a供体适配基因编辑系统。通过SSA活性报告实验初步证明了Gal4BD与SlugCas9、AsCas12a N-端融合对其打靶活性影响较小。通过HDR效率报告实验进行功能验证并优化供体设计方案，结果表明：对于CRISPR/Gal4BD-AsCas12a DAS，适配器结合序列(binding sequence，BS)与供体5′-端融合(BS-dsDNA)效果较好；对于CRISPR/Gal4BD-SlugCas9 DAS，则BS与供体3′-端融合(dsDNA-BS)较佳。最终，利用CRISPR/Gal4BD-SlugCas9 DAS对HEK293T细胞中的EMX1、NUDT5、AAVS1三个基因位点分别实现了24%、37%、31%的精准编辑，相比对照组得到了显著提高。本研究为供体适配基因编辑系统的进一步优化提供了参考和借鉴，为后续动物分子设计育种应用研究提供了新的基因编辑工具。

关键词: 基因编辑, 同源引导修复, 供体适配, 小型化Cas蛋白, 适配器

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

杨森, 马宝霞, 钱泓润, 崔婕妤, 张潇筠, 李利达, 魏泽辉, 张智英, 王建刚, 徐坤. 基于小型化Cas蛋白的CRISPR/Gal4BD-Cas供体适配基因编辑系统研究[J]. 遗传, 2024, 46(9): 716-726.

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.

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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

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

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

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