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