Detection and Modulation of DNA Translocations During Multi-Gene Genome Editing in T Cells

Bothmer A, Gareau KW, Abdulkerim HS, Buquicchio F, Cohen L, Viswanathan R, Zuris JA, Marco E, Fernandez CA, Myer VE, Cotta-Ramusino C.
Source: CRISPR J
Publication Date: (2020)
Issue: Jun;3(3): 177-187
Research Area:
Basic Research
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
4D-Nucleofector® X-Unit


For each treatment, 500,000 cells were nucleofected using the Lonza 4D-Nucleofector (AAF-1002B 4D Nucleofector Core unit, AAF-1002X 4D-Nucleofector X unit, P2 Primary Cell 4D-Nucleofector V4XP-2012) using the CA-137 pulse code program. The dose for each RNP is indicated in Supplementary Table S2. After nucleofection, the cells were incubated at room temperature for 10 min and then resuspended in complete expansion medium (see Supplementary Table S3) before transferring to 96-well plates containing prewarmed medium. The cells were collected at the indicated time points post nucleofection for genomic DNA (gDNA) extraction. For the HBB8/B2M12 time course, T cells were restimulated with Dynabeads Human T-Activator CD3/CD28 in stimulation medium 3 days after nucleofection and transferred to expansion medium 5 days after nucleofection.


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