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

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

Abstract

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

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.

Figures/Tables 8

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

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

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

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

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

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

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

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

References 18

[1]	Larson RC, Maus MV. Recent advances and discoveries in the mechanisms and functions of CAR T cells[J]. Nat Rev Cancer, 2021, 21(3): 145-161. doi: 10.1038/s41568-020-00323-z pmid: 33483715
[2]	Russo-Carbolante EM, Picanço-Castro V, Alves DCC, et al. Integration pattern of HIV-1 based lentiviral vector carrying recombinant coagulation factor VIII in Sk-Hep and 293T cells[J]. Biotechnol Lett, 2011, 33(1): 23-31. doi: 10.1007/s10529-010-0387-5 pmid: 20812025
[3]	Atianand MK, Fitzgerald KA. Molecular basis of DNA recognition in the immune system[J]. J Immunol, 2013, 190(5): 1911-1918. doi: 10.4049/jimmunol.1203162 pmid: 23417527
[4]	Monjezi R, Miskey C, Gogishvili T, et al. Enhanced CAR T-cell engineering using non-viral Sleeping Beauty transposition from minicircle vectors[J]. Leukemia, 2017, 31(1): 186-194. doi: 10.1038/leu.2016.180 pmid: 27491640
[5]	Foster JB, Choudhari N, Perazzelli J, et al. Purification of mRNA encoding chimeric antigen receptor is critical for generation of a robust T-cell response[J]. Hum Gene Ther, 2019, 30(2): 168-178. doi: 10.1089/hum.2018.145 pmid: 30024272
[6]	Kosicki M, Tomberg K, Bradley A. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements[J]. Nat Biotechnol, 2018, 36(8): 765-771. doi: 10.1038/nbt.4192 pmid: 30010673
[7]	Casini A, Olivieri M, Petris G, et al. A highly specific SpCas9 variant is identified by in vivo screening in yeast[J]. Nat Biotechnol, 2018, 36(3): 265-271.
[8]	Chen BH, Gilbert LA, Cimini BA, et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system[J]. Cell, 2013, 155(7): 1479-1491. doi: 10.1016/j.cell.2013.12.001 pmid: 24360272
[9]	Yu Y, Guo YJ, Tian QQ, et al. An efficient gene knock-in strategy using 5'-modified double-stranded DNA donors with short homology arms[J]. Nat Chem Biol, 2020, 16(4): 387-390. doi: 10.1038/s41589-019-0432-1 pmid: 31873222
[10]	Yin JH, Lu RS, Xin CC, et al. Cas9 exo-endonuclease eliminates chromosomal translocations during genome editing[J]. Nat Commun, 2022, 13(1): 1204. doi: 10.1038/s41467-022-28900-w pmid: 35260581
[11]	Yin JH, Fang KL, Gao YX, et al. Safeguarding genome integrity during gene-editing therapy in a mouse model of age-related macular degeneration[J]. Nat Commun, 2022, 13(1): 7867. doi: 10.1038/s41467-022-35640-4 pmid: 36550137
[12]	Kath J, Du WJ, Pruene A, et al. Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR Tcells[J]. Mol Ther Methods Clin Dev, 2022, 25: 311-330.
[13]	Liu XJ, Zhang YP, Cheng C, et al. CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells[J]. Cell Res, 2017, 27(1): 154-157. doi: 10.1038/cr.2016.142 pmid: 27910851
[14]	Yang M, Tkach D, Boyne A, et al. Optimized two-step electroporation process to achieve efficient nonviral-mediated gene insertion into primary T cells[J]. FEBS Open Bio, 2022, 12(1): 38-50.
[15]	Harrigan JA, Fan JS, Momand J, et al. WRN exonuclease activity is blocked by DNA termini harboring 3' obstructive groups[J]. Mech Ageing Dev, 2007, 128(3): 259-266. pmid: 17224176
[16]	Liao Q, Zheng JW, Wang B, et al. Efficient soluble expression and application of SpCas9 protein[J]. Food Science, 2023, 44(10): 150-157. doi: 10.7506/spkx1002-6630-20220429-391
[17]	刘芳, 卢婷, 蔡梦迪, 等. Cas9蛋白的克隆表达、分离纯化及多克隆抗体制备[J]. 贵州医科大学学报, 2019, 44(7): 757-761, 766.
	Liu F, Lu T, Cai MD, et al. Cloning expression, purification and polyclonal antibody preparation of Cas9 protein[J]. J Guizhou Med Univ, 2019, 44(7): 757-761, 766.
[18]	Hu JZ, Yin JX. Fusion protein and use method thereof: WO2023011638(A1)[P]. 2023-02-09.