CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell Therapy
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...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-06-01
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| Series: | Cancers |
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| Online Access: | https://www.mdpi.com/2072-6694/12/6/1704 |
| _version_ | 1797564100098654208 |
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| author | Zelda Odé Jose Condori Nicolas Peterson Sheng Zhou Giedre Krenciute |
| author_facet | Zelda Odé Jose Condori Nicolas Peterson Sheng Zhou Giedre Krenciute |
| author_sort | Zelda Odé |
| collection | DOAJ |
| description | 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 (<i>TRAC</i>) 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 <i>TRAC</i> or interleukin-13 (<i>IL-13</i>) 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. |
| first_indexed | 2024-03-10T18:52:38Z |
| format | Article |
| id | doaj.art-b9d0cab6c3d74dc9b11e1d961573a299 |
| institution | Directory Open Access Journal |
| issn | 2072-6694 |
| language | English |
| last_indexed | 2024-03-10T18:52:38Z |
| publishDate | 2020-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cancers |
| spelling | doaj.art-b9d0cab6c3d74dc9b11e1d961573a2992023-11-20T05:03:09ZengMDPI AGCancers2072-66942020-06-01126170410.3390/cancers12061704CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell TherapyZelda Odé0Jose Condori1Nicolas Peterson2Sheng Zhou3Giedre Krenciute4Department of Bone Marrow Transplantation and Cellular Therapy, St Jude Children’s Research Hospital, Memphis, TN 38105, USADepartment of Bone Marrow Transplantation and Cellular Therapy, St Jude Children’s Research Hospital, Memphis, TN 38105, USAGraduate School of Biomedical Sciences, St Jude Children’s Research Hospital, Memphis, TN 38105, USAExperimental Cellular Therapeutics Lab, St Jude Children’s Research Hospital, Memphis, TN 38105, USADepartment of Bone Marrow Transplantation and Cellular Therapy, St Jude Children’s Research Hospital, Memphis, TN 38105, USAT 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 (<i>TRAC</i>) 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 <i>TRAC</i> or interleukin-13 (<i>IL-13</i>) 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.https://www.mdpi.com/2072-6694/12/6/1704CART cellnon-viralHDRCRISPR-Cas9knock-in |
| spellingShingle | Zelda Odé Jose Condori Nicolas Peterson Sheng Zhou Giedre Krenciute CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell Therapy Cancers CAR T cell non-viral HDR CRISPR-Cas9 knock-in |
| title | CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell Therapy |
| title_full | CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell Therapy |
| title_fullStr | CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell Therapy |
| title_full_unstemmed | CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell Therapy |
| title_short | CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell Therapy |
| title_sort | crispr mediated non viral site specific gene integration and expression in t cells protocol and application for t cell therapy |
| topic | CAR T cell non-viral HDR CRISPR-Cas9 knock-in |
| url | https://www.mdpi.com/2072-6694/12/6/1704 |
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