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Open AccessArticle

CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell Therapy

1
Department of Bone Marrow Transplantation and Cellular Therapy, St Jude Children’s Research Hospital, Memphis, TN 38105, USA
2
Graduate School of Biomedical Sciences, St Jude Children’s Research Hospital, Memphis, TN 38105, USA
3
Experimental Cellular Therapeutics Lab, St Jude Children’s Research Hospital, Memphis, TN 38105, USA
*
Author to whom correspondence should be addressed.
Cancers 2020, 12(6), 1704; https://doi.org/10.3390/cancers12061704
Received: 30 April 2020 / Revised: 22 June 2020 / Accepted: 24 June 2020 / Published: 26 June 2020
T cells engineered with chimeric antigen receptors (CARs) show great promise in the treatment of some cancers. Modifying T cells to express CARs generally relies on T-cell transduction using viral vectors carrying a transgene, resulting in semi-random DNA integration within the T-cell genome. While this approach has proven successful and is used in generating the Food and Drug Administration (FDA, USA) approved B-lymphocyte antigen CD19-specific CAR T cells, it is possible the transgene could integrate into a locus that would lead to malignant transformation of the engineered T cells. In addition, manufacturing viral vectors is time-consuming and expensive. One way to overcome these challenges is site-specific gene integration, which can be achieved through clustered regularly interspaced short palindromic repeat (CRISPR) mediated editing and non-viral DNA, which serves as a template for homology-directed repair (HDR). This non-viral gene editing approach provides a rapid, highly specific, and inexpensive way to engineer T cells. Here, we describe an optimized protocol for the site-specific knock-in of a large transgene in primary human T cells using non-viral double stranded DNA as a repair template. As proof-of-principle, we targeted the T-cell receptor alpha constant (TRAC) locus for insertion of a large transgene containing green fluorescence protein (GFP) and interleukin-15 (IL-15). To optimize the knock-in conditions we tested template DNA concentration, homology arm length, cell number, and knock-in efficiency over time. We then applied these established guidelines to target the TRAC or interleukin-13 (IL-13) locus for the knock-in of synthetic molecules, such as a CAR, bispecific T-cell engager (BiTE), and other transgenes. While integration efficiency depends on the targeted gene locus and selected transgene, this optimized protocol reliably generates the desired insertion at rates upwards of 20%. Thus, it should serve as a good starting point for investigators who are interested in knocking in transgenes into specific loci. View Full-Text
Keywords: CAR; T cell; non-viral; HDR; CRISPR-Cas9; knock-in; TRAC CAR; T cell; non-viral; HDR; CRISPR-Cas9; knock-in; TRAC
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MDPI and ACS Style

Odé, Z.; Condori, J.; Peterson, N.; Zhou, S.; Krenciute, G. CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell Therapy. Cancers 2020, 12, 1704.

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