Frequent Aneuploidy in Primary Human T Cells Following CRISPR-Cas9 cleavage

Abstract

SUMMARYMultiple ongoing clinical trials use site-specific nucleases to disrupt T cell receptor (TCR) genes in order to allow for allogeneic T cell therapy1–5. 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 patients6. 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.

Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing

“…4e ). Although nuclease approaches have achieved targeted sequence inversions previously 49 , 50 , DSB-induced repair pathways generate undesired products and can lead to de-novo structural variants, including deletion of the targeted DNA sequence 20 - 22 and chromosomal abnormalities 23 , 24 . TwinPE and Bxb1 achieves comparable efficiency while circumventing the uncontrolled nature of DSB repair.…”

“…The use of paired nucleases for targeted deletion generates multiple byproducts 13 , 19 , and the precise location of the deletions is restricted by PAM availability. Moreover, DSBs at on-target or off-target sites can promote large deletions 20 - 22 , chromosomal abnormalities 23 , 24 , and chromothripsis 25 . The tendency of DSBs to generate complex mixtures of undesired byproducts and chromosomal changes 26 - 28 poses considerable challenges when applying nuclease-based editing for the manipulation of larger DNA sequences, especially in therapeutic settings.…”

CAR-NK cell therapy for glioblastoma: what to do next?

“…Although the efficiency of electroporation is high, it will reduce cell viability, and the genetic modification of electroporation is transient. Despite this, there have been many successful reports on electroporation technology in CAR-T cells preparation ( 131 ) ( 132 ) ( 133 ). Therefore, electroporation technology will also make a breakthrough in CAR-NK cells.…”

Nonviral base editing ofKCNJ13mutation preserves vision in an inherited retinal channelopathy

“…However, this strategy would require precise correction of a single nucleotide, which is difficult to achieve in post-mitotic cells using CRISPR-Cas9 nucleases followed by either homology-directed repair (HDR) or non-homologous end joining (NHEJ) (13)(14)(15). Moreover, double-strand DNA break (DSB) formation at the target site would pose a significant risk of uncontrolled indels as well as other undesired cellular consequences like chromosomal translocations, large deletions, aneuploidy, p53 activation, chromothripsis, transposon insertions (16)(17)(18)(19)(20)(21)(22)(23)(24)(25). Base editing, on the other hand, is poised to overcome these limitations by capitalizing on the RNA-guided programmability of CRISPR-Cas9 to deliver a base modification enzyme that can site-specifically convert a single nucleotide to another (26).…”