利用Cas9TX实现非病毒TRAC定点整合制备T细胞

doi:10.13560/j.cnki.biotech.bull.1985.2024-0321

摘要/Abstract

摘要：

【目的】Cas9TX是Cas9的变体，能够显著降低基因编辑过程中染色体易位，大幅提升基因编辑的安全性。研究Cas9TX替换Cas9实现基因定点敲入的可行性。【方法】首先，利用His标签蛋白纯化技术制备Cas9TX蛋白，并在细胞水平上通过绘制EC50和核酸酶裂解动力学曲线等进行功能验证。其次，以RNP和dsDNA混合物电转的方式编辑体外激活的T细胞，流式检测定点敲入的效率。最后，探讨了供体模板进行稳定性修饰提升定点敲入效率的可行性。【结果】制备的Cas9TX在T细胞TRAC基因的A、R和S靶点的敲除效率分别为71.8%、81.0%和79.9%，具有与Cas9相当的基因敲除效率。在TRAC 1号外显子分别设计的dsDNA供体模板（2A-GFP编码序列）和ssDNA供体模板（+ GTC bp），发现Cas9TX RNP定点敲入两种模板的效率均显著低于Cas9 RNP。通过DNA修饰制备防TREX2核酸外切酶降解的供体模板，不能提升Cas9TX RNP 定点敲入的效率。【结论】Cas9TX RNP在TRAC三个靶点的基因敲除方面可以替代Cas9 RNP使用，但定点整合外源基因的效率约为Cas9 RNP的一半。为Cas9TX应用于基因定点敲入提供了重要参考。

关键词: Cas9TX, 定点整合, RNP, dsDNA, CRISPR/Cas9

Abstract:

【Objective】Cas9TX, a Cas9 exo-endonuclease, can significantly reduce the chromosomal translocation and greatly improve the safety of gene editing. Thus this work aims to study the feasibility of Cas9TX replacing Cas9 for gene loci knockin.【Method】 Firstly, His-tagged Cas9TX protein was prepared and purified. Exo-endonuclease function of Cas9TX was verified using the curves of EC50 and kinetics of nuclease cleavage. Secondly, in vitro activated human T cells were edited via electroporating with Cas9TX RNP and dsDNA. The knockin efficiencies were measured by FACS. Finally, the feasibility of stability-modifications to donor templates for enhancing site-directed knock-in efficiency was explored. 【Result】 The knock-out efficiencies of the prepared Cas9TX at three TRAC-targeted site A, R and S of the T cells were 71.8%, 81.0% and 79.9%, respectively, which were equivalent to that by Cas9. However, the efficiencies of targeted integration of 2A-GFP（dsDNA donor templates designed）or +GTC bp（ssDNA donor templates）into the first exon of TRAC by Cas9TX were much lower than that by Cas9. DNA modifications that inhibit TREX2 exonuclease digestion could not improve the target knock-in efficiency of Cas9TX RNP. 【Conclusion】 Here we uncovered that Cas9TX RNP may be used for replacement of Cas9 RNP in the gene knock-out in three targets of TRAC，while the targeted integration efficiency using Cas9TX RNP is about half of using Cas9 RNP. This work serves as a valuable reference for using Cas9TX in targeted knock-in strategies.

Key words: Cas9TX, knock in, RNP, dsDNA, CRISPR/Cas9

崔海洋, 谭淼, 全壮, 陈红利, 董艳敏, 唐立春. 利用Cas9TX实现非病毒TRAC定点整合制备T细胞[J]. 生物技术通报, 2024, 40(9): 190-197.

CUI Hai-yang, TAN Miao, QUAN Zhuang, CHEN Hong-li, DONG Yan-min, TANG Li-chun. Generation of Virus-free TRAC-knocked-in T Cells Using Cas9TX[J]. Biotechnology Bulletin, 2024, 40(9): 190-197.

图/表 8

表1 序列信息

Table 1 Sequences information

名称Name		序列Sequence（5'-3'）
PCR Primers^[13]	TRAC-F4	ATCACGAGCAGCTGGTTTCT
PCR Primers^[13]	TRAC-R3	GCCACCTTCTCTTCATCTGC
qPCR Primers^[14]	TRAC-Up-F	GCATTTCAGGTTTCCTTGAGTGGCAG
	TRAC-Up-R	TGGCAAGTCACGGTCTCATGCTTTAT
	TRAC-Cross-F	CTTGTCCATCACTGGCATCTGGACTC
	TRAC-Cross-R	ATCGGTGTGAATAGGCAGACAGACTTGT
gRNAs^[13]	TRAC-A	ACAGATATCCAGAACCCTG
	TRAC-R	TGTACCAGCTGAGAGACTCT
	TRAC-S	ACAAAACTGTGCTAGACATG
ssDNA Templates^[15]	ssODN-F/ssODN-3'P	AGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGTCGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGC
ssDNA Templates^[15]	ssODN-R/ssODN-R-3'P	GCTCCAGGCCACAGCACTGTTGCTCTTGAAGTCCATAGACCTCATGTCGACTAGCACAGTTTTGTCTGTGATATACACATCAGAATCCTTACT

表2 RNP体外酶切反应体系

Table 2 Enzymatic digestion reaction system of RNP in vitro

组分Components	用量Volume/μL
Nuclease Free ddH₂O	19.8
NEB Buffer 2.1	2.5
TRAC DNA Template	2
RNP	0.7
Total	25

图1 SDS-PAGE检测Cas9和Cas9TX蛋白 1-2: Cas9TX 裂解液上清；3-4: Cas9 裂解液上清；5: 纯化的 Cas9 蛋白；6: 纯化的Cas9TX 蛋白

Fig. 1 Detection of Cas9 and Cas9TX protein by SDS-PAGE 1-2: Supernatant of Cas9TX bacterial lysate;3-4: supernatant of Cas9 bacterial lysate; 5: purified Cas9 protein; 6: purified Cas9TX protein

图2 Cas9和Cas9TX 体外酶切活性鉴定 A: RNP体外酶切目的片段. 1: 目的片段; 2-3: Cas9TX RNP+目的片段; 4-5: Cas9 RNP +目的片段; B: 不同浓度Cas9TX RNP切割目的片段. 1: 0.1 μmol/L; 2: 0.3 μmol/L; 3: 0.5 μmol/L; 4: 0.7 μmol/L; 5: 0.9 μmol/L

Fig. 2 Characterization of nuclease activity of Cas9 and Cas9TX in vitro A: RNP digestion of the TRAC DNA fragment. 1: target fragment; 2-3: target fragment with Cas9TX RNP; 4-5: target fragment with Cas9 RNP. B: Cut fragment with different concentrations of Cas9TX RNP. 1: 0.1 μmol/L; 2: 0.3 μmol/L; 3: 0.5 μmol/L; 4: 0.7 μmol/L; 5: 0.9 μmol/L

图3 T细胞内Cas9和Cas9TX的酶活性比较 A: TRAC gRNA靶点示意图；B: qPCR方案示意图；C：EC50 曲线；D: DSB生成曲线

Fig. 3 Comparison of enzyme activity of Cas9 and Cas9TX in T cell A: gRNA targeted sites. B: Scheme of qPCR strategy to measure unlinked DSBs. C: EC50 curve. D: Kinetics curve of DSB generation

图4 Cas9和Cas9TX在TRAC不同靶点的编辑效率 A：TIDE 分析Cas9在TRAC-S的敲除效率；B：TIDE分析Cas9TX 在TRAC-S的敲除效率；C：不同靶点敲除效率的统计结果

Fig. 4 Editing efficiencies of Cas9 and Cas9TX in different TRAC targets A: Knock out efficiency of Cas9 at TRAC-S site. B: Knock out efficiency of Cas9TX at TRAC-S site. C: Summary of knock out efficiencies at different targeted sites

图5 Cas9TX和Cas9TX RNP可降解供体模板

Fig. 5 DNA donor templates can be degraded by Cas9TX and Cas9TX RNP A: dsDNA; B: ssODN; C: GW dsDNA; D: 3' P ssODN. 1, donor template; 2, donor template with Cas9; 3, donor template with Cas9TX; 4, donor template with Cas9 RNP; 5, donor template with Cas9TX RNP

图6 供体模板稳定性修饰对定点敲入效率的影响 A：TRAC基因定点敲入方案模式图；B：流式分析Cas9组GFP表达比例；C：流式分析Cas9TX组GFP表达比例；D：GFP表达比例统计结果；E：TIDE分析Cas9组GTC碱基插入比例； F：TIDE分析Cas9TX组GTC碱基插入比例；G：GTC插入比例统计结果

Fig. 6 Effects of donor-template modification on the site-directed insertion efficiencies A: Scheme of 2A-GFP or GTC knock in strategies; B: flow cytometric analysis for GFP expression using Cas9; C: flow cytometric analysis for GFP expression using Cas9TX; D: statistical results of GFP knock in efficiencies; E: TIDE analysis for GTC knock in efficiencies using Cas9; F: TIDE analysis for GTC knock in efficiencies using Cas9TX;G: statistical results of GTC knock in efficiencies

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