Controlling DNA-deletionsizes in CRISPR-edited human T cells by the strength of TCR stimulation and pifithrin-a
CRISPR/Cas9-engineered human T cells hold great promise in improving T cell effector functions for adoptive T cell therapies. To ensure safety during CRISPR/Cas9-editing optimized gRNAs, novel variants or prediction tools for indel outcomes have been developed. Still, several reports describe the occurrence of chromosomal truncations or loss of large DNA fragments of up to several kbs after CRISPR/Cas9 editing. So far, the measures to increase the safety of T cell products focused on the CRISPR Cas9 system. However, T cell-intrinsic features such as their massive expansion after TCR stimulation have not been taken into consideration. Here, we describe driving forces of indel formation in primary human T cells. Increased T cell activation and proliferation speed correlate with higher knock-out rates and larger deletions. Editing of non-activated T cells reduces the risk of unwanted large deletions but with the downside of reduced knock-out efficiencies. An alternative strategy to reduce the risk of large deletions is the addition of the small molecule pifithrin-a after CRISPR/Cas9 editing. Pifithrin-a treatment results in a smaller, more defined deletion pattern. PFT-a furthermore reduces the risk of unwanted chromosomal translocations and aneuploidy while maintaining the functionality of CRISPR-engineered T cells. Controlling T cell activation and the addition of pifithrin-a are easily accessible strategies for safer CRISPR/Cas9-engineering for adoptive T cell therapies. Overall design: CD4 and PDCD1 gene loci were simultaneously targeted with Cas9 RNPs in activated and non-activated T cells. Genotoxicity was assesed via CAST-Seq.