CRISPR/Cas9-induced structural variations expand in T lymphocytes in vivo

Abstract

CRISPR/Cas9 has been adapted to disrupt endogenous genes in adoptive T-lymphocyte therapy to prevent graft-versus-host disease. However, genome editing also generates prevalent deleterious structural variations (SVs), including chromosomal translocations and large deletions, raising safety concerns about reinfused T cells. Here, we dynamically monitored the progression of SVs in a mouse model of T-cell receptor (TCR)-transgenic T-cell adoptive transfer, mimicking TCR T therapeutics. Remarkably, CRISPR/Cas9-induced SVs persist and undergo clonal expansion in vivo after three weeks or even two months, evidenced by high enrichment and low junctional diversity of identified SVs post infusion. Specifically, we detected 128 expanded translocations, with 20 615 as the highest number of amplicons. The identified SVs are stochastically selected among different individuals and show an inconspicuous locus preference. Similar to SVs, viral DNA integrations are routinely detected in edited T cells and also undergo clonal expansion. The persistent SVs and viral DNA integrations in the infused T cells may constantly threaten genome integrity, drawing immediate attention to the safety of CRISPR/Cas9-engineered T cells mediated immunotherapy.

CRISPR/Cas9 Gene Editing System Can Alter Gene Expression and Induce DNA Damage Accumulation

“…Gene editing using CRISPR/Cas9 induces DSBs and various other catastrophic events, such as genomic rearrangements and chromosomal variations [ 10 , 17 , 18 ]. Clinical trials involving CRISPR-Cas9 have emphasized that safety is a limitation of its applications [ 19 , 20 , 21 ].…”

“…Therefore, attention should be paid to AS events when performing gene editing using CRISPR/Cas9 that can generate novel proteins, some of which may be highly oncogenic. Moreover, CRISPR has been reported to trigger changes in the chromosomal structure [ 10 , 17 ]. A fusion gene is a chimeric gene in which all or part of the sequences of two genes are fused, generally caused by chromosomal translocation or deletion.…”

“…Aneuploidy and chromosomal truncations occur frequently in human T cells upon CRISPR/Cas9 cleavage using clinical gRNA sequences [ 16 ]. Importantly, Wu et al [ 17 ] found that the chromosomal translocations, large-scale loss of chromosomes, and viral DNA insertions caused by gene editing do not disappear with time and continue to remain at high levels, showing a clear random clonal expansion. Höijer et al [ 18 ] found that both the first and second generations of zebrafish had structural variations in the edited fertilized eggs, and these variations were both on- and off-target.…”

Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing

“…These events exploit the availability of frequent DSBs generated by CRISPR–Cas9 nuclease to favor the insertion of exogenous DNA fragments using microhomology-mediated end joining (MMEJ) 25 , 57 , 92 , 96 . Although chromosomal rearrangements are likely rare events, the concern is that even a very small number of CRISPR–Cas9-edited cells harboring these detrimental events may clonally expand in vivo 97 and cause diseases.…”

Recent advances and applications of CRISPR-Cas9 in cancer immunotherapy

“…NHEJ and MMEJ typically causes INDELs of genes to disrupt protein-coding sequences and develops functional knockouts, but may generate deleterious DSB repair byproducts, including cancer-driving chromosomal translocations, large chromatin deletions, and vector insertions in humans. The structural variations (SVs) has become another dimension of threat to genome stability during genome editing [ 186 , 187 ]. PEM-seq, HTGTS and SuperQ were explored to distinguish various DNA repair products induced by CRISPR–Cas9.…”