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

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

不同CRISPR/Cas9供体适配基因编辑系统的比较及优化研究

马宝霞¹(), 杨森¹, 吕明¹, 王昱人¹, 常立业¹, 韩艺帆¹, 王建刚¹, 郭杨²(), 徐坤¹()

1.西北农林科技大学动物科技学院，杨凌 712100
2.新疆维吾尔自治区畜牧总站，乌鲁木齐 830002

收稿日期:2024-01-07 修回日期:2024-03-22 出版日期:2024-06-20 发布日期:2024-03-27
通讯作者: 郭杨，硕士，高级畜牧师，研究方向：动物遗传育种。E-mail: guoyang302@163.com;徐坤，博士，副教授，研究方向：动物生物技术。E-mail: xukunas@nwafu.edu.cn
作者简介:马宝霞，硕士研究生，专业方向：动物基因编辑技术。E-mail: 18792898523@163.com
基金资助:
科技创新2030?农业生物育种重大项目(2023ZD04074);科技创新2030?农业生物育种重大项目(2023ZD04051)

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 Published:2024-06-20 Online: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)

摘要/Abstract

摘要：

在哺乳动物细胞中进行基因敲入通常采用同源定向修复(homology-directed repair, HDR)机制将外源DNA模板整合到目标基因组靶点中。然而HDR效率往往较低，其中外源DNA模板与目标基因组靶点的共定位是关键限制因素之一。为提高CRISPR/Cas9系统介导的HDR效率，本团队及前人研究将不同接头蛋白与SpCas9蛋白融合表达，利用其与特异性DNA序列结合的特性，构建了多种CRISPR/SpCas9供体适配基因编辑系统。为了便于比较、优化不同CRISPR/Cas9供体适配系统，本研究利用这些系统在HEK293T细胞GAPDH和ACTB基因末位外显子3′-端进行了eGFP基因敲入，并采用了优化的供体DNA模板设计方式，通过PCR和Sanger测序检测敲入的准确性，流式细胞分析进行敲入效率的检测。结果表明，将yGal4BD、hGal4BD、hLacI、hTHAP11和N57等接头蛋白与SpCas9蛋白C-端融合对其活性均无显著性影响；在GAPDH位点上，SpCas9融合yGal4BD、hGal4BD、hLacI和hTHAP11的供体适配系统等均能显著提高敲入效率；在ACTB位点上，SpCas9融合yGal4BD和hGal4BD能显著提高敲入效率；且增加供体DNA模板中的结合序列(binding sequence, BS)数量，有利于提高SpCas9-hTHAP11系统介导的敲入效率。总之，本研究比较并优化了不同的CRISPR/Cas9供体适配基因编辑系统，为后续相关的基因编辑应用研究提供了参考和借鉴。

关键词: 基因编辑, 基因敲入, CRISPR/Cas9, 供体适配, 同源定向修复

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

马宝霞, 杨森, 吕明, 王昱人, 常立业, 韩艺帆, 王建刚, 郭杨, 徐坤. 不同CRISPR/Cas9供体适配基因编辑系统的比较及优化研究[J]. 遗传, 2024, 46(6): 466-477.

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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参考文献 26

[1]	Zhang HX, Zhang Y, Yin H. Genome editing with mRNA encoding ZFN, TALEN, and Cas9. Mol Ther, 2019, 27(4): 735-746.
[2]	Carroll D. Genome engineering with targetable nucleases. Annu Rev Biochem, 2014, 83: 409-439. doi: 10.1146/annurev-biochem-060713-035418 pmid: 24606144
[3]	Shalem O, Sanjana NE, Zhang F. High-throughput functional genomics using CRISPR-Cas9. Nat Rev Genet, 2015, 16(5): 299-311. doi: 10.1038/nrg3899 pmid: 25854182
[4]	Wang JY, Doudna JA. CRISPR technology: a decade of genome editing is only the beginning. Science, 2023, 379(6629): eadd8643.
[5]	Fu YW, Dai XY, Wang WT, Yang ZX, Zhao JJ, Zhang JP, Wen W, Zhang F, Oberg KC, Zhang L, Cheng T, Zhang XB. Dynamics and competition of CRISPR-Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing. Nucleic Acids Res, 2021, 49(2): 969-985. doi: 10.1093/nar/gkaa1251 pmid: 33398341
[6]	Rees HA, Liu DR. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat Rev Genet, 2018, 19(12): 770-788.
[7]	Anzalone AV, Koblan LW, Liu DR. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat Biotechnol, 2020, 38(7): 824-844. doi: 10.1038/s41587-020-0561-9 pmid: 32572269
[8]	Mikkelsen NS, Bak RO. Enrichment strategies to enhance genome editing. J Biomed Sci, 2023, 30(1): 51. doi: 10.1186/s12929-023-00943-1 pmid: 37393268
[9]	Yang H, Ren SL, Yu SY, Pan HF, Li TD, Ge SX, Zhang J, Xia NS. Methods favoring homology-directed repair choice in response to CRISPR/Cas9 induced-double strand breaks, Int J Mol Sci, 2020, 21(18): 6461.
[10]	Sun WL, Liu H, Yin WH, Qiao J, Zhao XK, Liu Y. Strategies for enhancing the homology-directed repair efficiency of CRISPR-Cas systems. CRISPR J, 2022, 5(1): 7-18.
[11]	Shy BR, Vykunta VS, Ha A, Talbot A, Roth TL, Nguyen DN, Pfeifer WG, Chen YY, Blaeschke F, Shifrut E, Vedova S, Mamedov MR, Chung JYJ, Li H, Yu R, Wu D, Wolf J, Martin TG, Castro CE, Ye L, Esensten JH, Eyquem J, Marson A. High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails. Nat Biotechnol, 2023, 41(4): 521-531.
[12]	Riesenberg S, Kanis P, Macak D, Wollny D, Düsterhöft D, Kowalewski J, Helmbrecht N, Maricic T, Pääbo S. Efficient high-precision homology-directed repair-dependent genome editing by HDRobust. Nat Methods, 2023, 20(9): 1388-1399. doi: 10.1038/s41592-023-01949-1 pmid: 37474806
[13]	Ma M, Zhuang FF, Hu XB, Wang BL, Wen XZ, Ji JF, Xi JZJ. Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-Avidin/Biotin-donor DNA system. Cell Res, 2017, 27(4): 578-581. doi: 10.1038/cr.2017.29 pmid: 28266543
[14]	Savic N, Ringnalda FC, Lindsay H, Berk C, Bargsten K, Li YZ, Neri D, Robinson MD, Ciaudo C, Hall J, Jinek M, Schwank G. Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology- directed repair. eLife, 2018, 7: e33761.
[15]	Gu B, Posfai E, Rossant J. Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos. Nat Biotechnol, 2018, 36(7): 632-637. doi: 10.1038/nbt.4166 pmid: 29889212
[16]	Aird EJ, Lovendahl KN, St Martin A, Harris RS, Gordon WR. Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template. Commun Biol, 2018, 1: 54. doi: 10.1038/s42003-018-0054-2 pmid: 30271937
[17]	Lee K, Mackley VA, Rao A, Chong AT, Dewitt MA, Corn JE, Murthy N. Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering. eLife, 2017, 6: e25312.
[18]	Li GL, Wang HQ, Zhang XW, Wu ZF, Yang HQ. A Cas9-transcription factor fusion protein enhances homology-directed repair efficiency. J Biol Chem, 2021, 296: 100525.
[19]	Ma SF, Wang XL, Hu YF, Lv J, Liu CF, Liao KT, Guo XH, Wang D, Lin Y, Rong ZL. Enhancing site-specific DNA integration by a Cas9 nuclease fused with a DNA donor-binding domain. Nucleic Acids Res, 2020, 48(18): 10590-10601. doi: 10.1093/nar/gkaa779 pmid: 32986839
[20]	Wienert B, Nguyen DN, Guenther A, Feng SJ, Locke MN, Wyman SK, Shin J, Kazane KR, Gregory GL, Carter MAM, Wright F, Conklin BR, Marson A, Richardson CD, Corn JE. Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair. Nat Commun, 2020, 11(1): 2109. doi: 10.1038/s41467-020-15845-1 pmid: 32355159
[21]	Zhang XJ, Xu K, Shen JC, Mu L, Qian HR, Cui JY, Ma BX, Chen ZL, Zhang ZY, Wei ZH. A CRISPR/Cas9- Gal4BD donor adapting system for enhancing homology- directed repair. Hereditas (Beijing), 2022, 44(8): 708-719.
	张潇筠, 徐坤, 沈俊岑, 穆璐, 钱泓润, 崔婕妤, 马宝霞, 陈知龙, 张智英, 魏泽辉. 一种新型提高HDR效率的CRISPR/Cas9-Gal4BD供体适配基因编辑系统. 遗传, 2022, 44(8): 708-719.
[22]	Ma BX, Cui JY, Qian HR, Zhang XJ, Yang S, Zhang QJ, Han YF, Zhang ZY, Wang JG, Xu K. A novel CRISPR/ Cas9-hLacI donor adapting system for dsDNA-templated gene editing. Chin J Biotechnol, 2023, 39(10): 4204-4218.
	马宝霞, 崔婕妤, 钱泓润, 张潇筠, 杨森, 张骐镜, 韩艺帆, 张智英, 王建刚, 徐坤. 一种新型的CRISPR/Cas9- hLacI双链DNA供体适配基因编辑系统. 生物工程学报, 2023, 39(10): 4204-4218.
[23]	Kong XZ, Yin RH, Ning HM, Zheng WW, Dong XM, Yang Y, Xu FF, Li JJ, Zhan YQ, Yu M, Ge CH, Zhang JH, Chen H, Li CY, Yang XM. Effects of THAP11 on erythroid differentiation and megakaryocytic differentiation of K562 cells. PloS One, 2014, 9(3): e91557.
[24]	Hew BE, Sato R, Mauro D, Stoytchev I, Owens JB. RNA-guided piggyBac transposition in human cells. Synth Biol (Oxf), 2019, 4(1): ysz018.
[25]	Xu K, Ren CH, Liu ZT, Zhang T, Zhang TT, Li D, Wang L, Yan Q, Guo LJ, Shen JC, Zhang ZY. Efficient genome engineering in eukaryotes using Cas9 from Streptococcus thermophiles. Cell Mol Life Sci, 2015, 72(2): 383-399.
[26]	Koch B, Nijmeijer B, Kueblbeck M, Cai Y, Walther N, Ellenberg J. Generation and validation of homozygous fluorescent knock-in cells using CRISPR-Cas9 genome editing. Nat Protoc, 2018, 13(6): 1465-1487. doi: 10.1038/nprot.2018.042 pmid: 29844520

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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

不同CRISPR/Cas9供体适配基因编辑系统的比较及优化研究

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

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