Unintended CRISPR-Cas9 editing outcomes: a review of the detection and prevalence of structural variants generated by gene-editing in human cells

Abstract

Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) gene-editing system (CRISPR-Cas) is a valuable tool for fundamental and applied research applications. Significant improvements in editing efficacy have advanced genome editing strategies into phase 3 human clinical trials. However, recent studies suggest that our understanding of editing outcomes has lagged behind the developments made in generating the edits themselves. While many researchers have analyzed on- and off-target events through the lens of small insertions or deletions at predicted sites, screens for larger structural variants (SVs) and chromosomal abnormalities are not routinely performed. Full and comprehensive validation of on- and off-target effects is required to ensure reproducibility and to accurately assess the safety of future editing applications. Here we review SVs associated with CRISPR-editing in cells of human origin and highlight the methods used to detect and avoid them.

Near-perfect precise on-target editing of human hematopoietic stem and progenitor cells

“…To address this issue, we demonstrated that by delivering a combination of silent and mutant donors (of the same strand to prevent donor annealing) one can re-introduce heterozygosity based on the ratio of donors delivered, allowing editing levels and zygosity to be tuned. Another consideration that would be important for any clinical use of edited cells is off-target editing and genomic rearrangements 29 . We did not observe any off-target edits at predicted sites, regardless of whether edits were done in the presence of AZD7648/p53 siRNA or not, suggesting that the protocol presented here did not substantially increase off-target editing frequencies.…”

Revolutionizing cancer treatment: enhancing CAR-T cell therapy with CRISPR/Cas9 gene editing technology

“…Investigations involving the editing of human embryonic stem cells and other cell types have revealed that the Cas9 protein can directly interact with the DNA-PK complex, impeding the DNA repair pathway and potentially heightening the risk of chromosome fragmentation ( 76 ). Moreover, the utilization of the CRISPR-Cas system has the potential to induce unforeseen structural variations (SVs), including deletions, duplications, and insertions ( 77 ). Consequently, CRISPR/Cas9 technology may raise some potential safety issues for CAR-T cell therapy that need to be carefully considered and addressed.…”

Monitoring Genomic Structural Rearrangements Resulting from Gene Editing

“…Fundamental to genome editing systems, such as clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated protein (Cas9), is the requisite induction of targeted DNA double-strand breaks (DSBs) to specific base sequences, and subsequent inactivation or replacement of the targeted or closely associated sequences [1][2][3][4]. However, because no genome editing technology or DNA repair pathway is 100% error-free, low-frequency but potentially genotoxic, structural variants are often observed in parallel with the desired edit(s) [5].…”

“…Targeted DSBs induced by guided endonucleases have an increased potential for mis-repair among broken DNA ends since they can be relatively close in time and proximity to other DSBs in the cell. Just as for DSBs induced by other sources, such as ionizing radiations, mis-repair events result in the creation of structural variants, including inversions, deletions, translocations, and even more complex chromosome aberrations [5,[8][9][10]. A simple rearrangement, such as an inversion, requires the mis-repair of two concurrent DSBs, while complex rearrangements involve three or more DSBs, and so the risk of formation of both simple and complex structural variants increases in direct proportion to the number of on-and off-target edits occurring in an individual cell.…”

“…The current interest in evaluating mis-repair or mis-rejoining events, structural variation, genotoxicity, and chromosomal/genomic instability in genome-edited populations has intensified due to observations of these events in CRISPR-Cas9 editing systems [5,10]. Although much of the focus has been on the induction of unintended off-target DSBs and their consequences, the potential genotoxic consequences of on-target DSBs are also an important outcome that require further exploration.…”