利用Cas9TX实现非病毒TRAC定点整合制备T细胞

Generation of Virus-free TRAC-knocked-in T Cells Using Cas9TX

【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Cas9RNP。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Cas9TXRNP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Cas9RNP使用,但定点整合外源基因的效率约为Cas9RNP的一半。为Cas9TX应用于基因定点敲入提供了重要参考。

【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,Histagged 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.