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Detection and Modulation of DNA Translocations During Multi-Gene Genome Editing in T Cells.
CRISPR J. 2020 Jun; 3(3):177-187.CJ

Abstract

Multiplexed genome editing with DNA endonucleases has broad application, including for cellular therapies, but chromosomal translocations, natural byproducts of inducing simultaneous genomic breaks, have not been explored in detail. Here we apply various CRISPR-Cas nucleases to edit the T cell receptor alpha and beta 2 microglobulin genes in human primary T cells and comprehensively evaluate the frequency and stability of the resulting translocations. A thorough translocation frequency analysis using three orthogonal methods (droplet digital PCR, unidirectional sequencing, and metaphase fluorescence in situ hybridization) yielded comparable results and an overall translocation rate of ∼7% between two simultaneous CRISPR-Cas9 induced edits. In addition, we show that chromosomal translocations can be reduced when using different nuclease combinations, or by the presence of a homologous single stranded oligo donor for multiplexed genome editing. Importantly, the two different approaches for translocation reduction are compatible with cell therapy applications.

Authors+Show Affiliations

Editas Medicine, Cambridge, Massachusetts, USA. Tessera Therapeutic, Cambridge, Massachusetts, USA.Editas Medicine, Cambridge, Massachusetts, USA.Editas Medicine, Cambridge, Massachusetts, USA. Third Rock NewCo, Cambridge, Massachusetts, USA.Editas Medicine, Cambridge, Massachusetts, USA. Program in Immunology, Stanford University, Stanford, California, USA. Department of Pathology, Stanford University, Stanford, California, USA.Editas Medicine, Cambridge, Massachusetts, USA. University of California San Diego, La Jolla, California, USA.Editas Medicine, Cambridge, Massachusetts, USA.Editas Medicine, Cambridge, Massachusetts, USA.Editas Medicine, Cambridge, Massachusetts, USA.Editas Medicine, Cambridge, Massachusetts, USA. Diagon Therapeutic, Boston Massachusetts, USA.Editas Medicine, Cambridge, Massachusetts, USA. Atlas Venture, Cambridge, Massachusetts, USA.Editas Medicine, Cambridge, Massachusetts, USA. Tessera Therapeutic, Cambridge, Massachusetts, USA.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

32584143

Citation

Bothmer, Anne, et al. "Detection and Modulation of DNA Translocations During Multi-Gene Genome Editing in T Cells." The CRISPR Journal, vol. 3, no. 3, 2020, pp. 177-187.
Bothmer A, Gareau KW, Abdulkerim HS, et al. Detection and Modulation of DNA Translocations During Multi-Gene Genome Editing in T Cells. CRISPR J. 2020;3(3):177-187.
Bothmer, A., Gareau, K. W., Abdulkerim, H. S., Buquicchio, F., Cohen, L., Viswanathan, R., Zuris, J. A., Marco, E., Fernandez, C. A., Myer, V. E., & Cotta-Ramusino, C. (2020). Detection and Modulation of DNA Translocations During Multi-Gene Genome Editing in T Cells. The CRISPR Journal, 3(3), 177-187. https://doi.org/10.1089/crispr.2019.0074
Bothmer A, et al. Detection and Modulation of DNA Translocations During Multi-Gene Genome Editing in T Cells. CRISPR J. 2020;3(3):177-187. PubMed PMID: 32584143.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Detection and Modulation of DNA Translocations During Multi-Gene Genome Editing in T Cells. AU - Bothmer,Anne, AU - Gareau,Kenneth W, AU - Abdulkerim,Hayat S, AU - Buquicchio,Frank, AU - Cohen,Lucas, AU - Viswanathan,Ramya, AU - Zuris,John A, AU - Marco,Eugenio, AU - Fernandez,Cecilia A, AU - Myer,Vic E, AU - Cotta-Ramusino,Cecilia, PY - 2020/6/26/entrez PY - 2020/6/26/pubmed PY - 2020/6/26/medline SP - 177 EP - 187 JF - The CRISPR journal JO - CRISPR J VL - 3 IS - 3 N2 - Multiplexed genome editing with DNA endonucleases has broad application, including for cellular therapies, but chromosomal translocations, natural byproducts of inducing simultaneous genomic breaks, have not been explored in detail. Here we apply various CRISPR-Cas nucleases to edit the T cell receptor alpha and beta 2 microglobulin genes in human primary T cells and comprehensively evaluate the frequency and stability of the resulting translocations. A thorough translocation frequency analysis using three orthogonal methods (droplet digital PCR, unidirectional sequencing, and metaphase fluorescence in situ hybridization) yielded comparable results and an overall translocation rate of ∼7% between two simultaneous CRISPR-Cas9 induced edits. In addition, we show that chromosomal translocations can be reduced when using different nuclease combinations, or by the presence of a homologous single stranded oligo donor for multiplexed genome editing. Importantly, the two different approaches for translocation reduction are compatible with cell therapy applications. SN - 2573-1602 UR - https://www.unboundmedicine.com/medline/citation/32584143/Detection_and_Modulation_of_DNA_Translocations_During_Multi-Gene_Genome_Editing_in_T_Cells L2 - https://www.liebertpub.com/doi/10.1089/crispr.2019.0074?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed DB - PRIME DP - Unbound Medicine ER -
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