Reprogramming Human T Cell Function and Specificity With Non–Viral Genome Targeting

Abstract

TL Roth, C Puig–Saus, R Yu. Nature. 2018;559(7714):405–409 To develop and optimize nonviral CRISPR-Cas9 genome editing for primary T cells and apply this technique in part to correct a pathogenic mutation in a monogenic autoimmune disease. Using CRISPR-Cas9, primary human T cells from healthy donors and a family with monogenic autoimmune disease were engineered ex vivo along with other important proofs of concept. The CRISPR-Cas9 system was first described as a prokaryotic adaptive immune response, and it has since been widely used as a gene editing tool. CRISPR sequences identify homologous areas in the DNA, and Cas9 creates precise double-stranded breaks. Double-stranded breaks are repaired by either nonhomologous end joining or homology-directed repair (HDR). HDR is less error prone, and by using CRISPR-Cas9 with HDR templates, transgenes can be integrated into the targeted genome with precision. In this study, an electroporation method was optimized for ex vivo primary human T cells to allow for uptake of CRISPR-Cas9 ribonucleoprotein complexes and large (>1 kb) double-stranded DNA (dsDNA) templates for HDR. The technique allowed for optimized cell viability along with efficiency under …