The CRISPR-Cas12a Platform for Accurate Genome Editing, Gene Disruption, and Efficient Transgene Integration in Human Immune Cells

CRISPR-Cas12a nucleases have expanded the toolbox for
targeted
genome engineering in a broad range of organisms. Here, using a high-throughput
engineering approach, we explored the potential of a novel CRISPR-MAD7
system for genome editing in human cells. We evaluated several thousand
optimization conditions and demonstrated accurate genome reprogramming
with modified MAD7. We identified crRNAs that allow for ≤95%
non-homologous end joining (NHEJ) and 66% frameshift mutations in
various genes and observed the high-cleavage fidelity of MAD7 resulting
in undetectable off-target activity. We explored the dsDNA delivery
efficiency of CRISPR-MAD7, and by using our optimized transfection
protocol, we obtained ≤85% chimeric antigen receptor (CAR)
insertions in primary T cells, thus exceeding the baseline integration
efficiencies of therapeutically relevant transgenes using currently
available virus-free technologies. Finally, we evaluated multiplex
editing efficiency with CRISPR-MAD7 and demonstrated simultaneous
≤35% CAR transgene insertions and ≤80% gene disruption
efficiencies. Both the platform and our transfection procedure are
easily adaptable for further preclinical studies and could potentially
be used for clinical manufacturing of CAR T cells.