Interrogating immune cells and cancer with CRISPR-Cas9.
Trends in immunology
CRISPR-Cas9 technologies have transformed the study of genetic pathways governing cellular differentiation and function. Recent advances have adapted these methods to immune cells, which has accelerated the pace of functional genomics in immunology and enabled new avenues for the design of cellular immunotherapies for cancer. In this review, we summarize recent developments in CRISPR-Cas9 technology and discuss how they have been leveraged to discover and manipulate novel genetic regulators of the immune system. We envision that these results will provide a valuable resource to aid in the design, implementation, and interpretation of CRISPR-Cas9-based screens in immunology and immuno-oncology.
View details for DOI 10.1016/j.it.2021.03.003
View details for PubMedID 33812776
Detection and Modulation of DNA Translocations During Multi-Gene Genome Editing in T Cells.
The CRISPR journal
2020; 3 (3): 177–87
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.
View details for DOI 10.1089/crispr.2019.0074
View details for PubMedID 32584143