Frequent aneuploidy in primary human T cells after CRISPR–Cas9 cleavage

Abstract

Multiple ongoing clinical trials use site-specific nucleases to disrupt T cell receptor (TCR) genes in order to allow for allogeneic T cell therapy [1][2][3][4][5] . In particular, the first U.S. clinical trial using CRISPR-Cas9 entailed the targeted disruption of the TCR chains and programmed cell death protein 1 (PDCD1) in T cells of refractory cancer patients 6 . Here, we used the same guide RNA sequences and applied single-cell RNA sequencing (scRNAseq) to more than 7000 primary human T cells, transfected with CRISPR-Cas9. Four days post-transfection, we found a loss of chromosome 14, harboring the TCRα locus, in up to 9% of the cells, and a chromosome 14 gain in up to 1.4% of the cells. We further identified truncations of chromosome 7, harboring the TCRβ locus, in 9.9% of the cells. Loss of heterozygosity (LOH) was further validated using fluorescence in situ hybridization (FISH) and the temporal dynamics of cleavage and incomplete repair were monitored using digital droplet PCR (ddPCR). Aneuploidy was found among all T cell subsets and was associated with transcriptional signatures of reduced proliferation and metabolism as well as with induced p53 activation and cell death. We conclude that aneuploidy and chromosomal truncations are frequent outcomes of CRISPR-Cas9 cleavage in clinical protocols. Monitoring and minimizing these aberrant products is crucial for future applications of genome editing in T cell engineering and beyond.

Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression

“…Still, this risk will require additional monitoring when translated to clinical manufacturing procedures. 55 Such concerns would be especially relevant for any multiplexed editing design (e.g., VFC-CAR with simultaneous PD-1 knockout). Finally, additional work to track changes to phenotype over the course of manufacture, and at various time points in vivo , could further elucidate the nuances of cytotoxicity dynamics in RV-CAR products, TRAC -CAR products, and VFC-CAR products targeting other genomic insertion sites.…”

Choice of template delivery mitigates the genotoxic risk and adverse impact of editing in human hematopoietic stem cells

“…Until now, investigation of the potential source of genotoxicity during gene editing mainly focused on off-target nuclease activity and the occurrence of genomic rearrangements at the target site ( Adikusuma et al., 2018 ; Boutin et al., 2021 ; Kosicki et al., 2018 ; Lattanzi et al., 2021 ; Leibowitz et al., 2021 ; Nahmad et al., 2022 ; Turchiano et al., 2021 ). The genotoxic risk ascribed to DNA template delivery, its persistence, and its integration in edited cells ( Colella et al., 2018 ; Penaud-Budloo et al., 2018 ; Schnödt and Büning, 2017 ) remains poorly investigated, especially for HSPCs.…”

CRISPR nuclease off-target activity and mitigation strategies

“…While the off-target detection methods described above are most useful for identifying localized effects of DNA double-strand breaks (DSBs), larger scale off-target effects have been observed. These include gross chromosomal rearrangements such as translocations ( Bothmer et al, 2020 ; Stadtmauer et al, 2020 ; Samuelson et al, 2021 ), chromothripsis ( Leibowitz et al, 2021 ), and aneuploidy ( Amendola et al, 2022 ; Nahmad et al, 2022 ). Translocation events most often occur as a consequence of: 1) on-target cleavage and recombination with a homologous region of the genome ( Turchiano et al, 2021 ); 2) simultaneous cleavage at an on-target and off-target sequence ( Lattanzi et al, 2021 ); or 3) following multiple on-target cleavage events in multiplexed editing workflows ( Qasim et al, 2017 ; Bothmer et al, 2020 ; Stadtmauer et al, 2020 ; Samuelson et al, 2021 ; Diorio et al, 2022 ).…”