Cas9 exo-endonuclease eliminates chromosomal translocations during genome editing

Abstract

The mechanism underlying unwanted structural variations induced by CRISPR-Cas9 remains poorly understood, and no effective strategy is available to inhibit the generation of these byproducts. Here we find that the generation of a high level of translocations is dependent on repeated cleavage at the Cas9-targeting sites. Therefore, we employ a strategy in which Cas9 is fused with optimized TREX2 to generate Cas9TX, a Cas9 exo-endonuclease, which prevents perfect DNA repair and thereby avoids repeated cleavage. In comparison with CRISPR-Cas9, CRISPR-Cas9TX greatly suppressed translocation levels and enhanced the editing efficiency of single-site editing. The number of large deletions associated with Cas9TX was also reduced to very low level. The application of CRISPR-Cas9TX for multiplex gene editing in chimeric antigen receptor T cells nearly eliminated deleterious chromosomal translocations. We report the mechanism underlying translocations induced by Cas9, and propose a general strategy for reducing chromosomal abnormalities induced by CRISPR-RNA-guided endonucleases.

Target residence of Cas9-sgRNA influences DNA double-strand break repair pathway choices in CRISPR/Cas9 genome editing

“…Previous studies have demonstrated that c-NHEJ is intrinsically accurate for these ends [ 6 , 7 , 29 ]. In each round of repair during CRISPR/Cas9 genome editing, about a half of NHEJ products are accurate in repair of Cas9-induced DSBs and the remaining half generate indels [ 6 , 8 , 9 ]. Thus, inactivation of c-NHEJ would increase the use of a-EJ in each round of CRISPR/Cas9 genome editing.…”

“…We then wondered what the difference is. While c-NHEJ of both I-SceI- and Cas9-induced DSBs generates a significant level of accurate end-joining products in each round of repair at their respective targets, regenerating the target sites for recleavage, the recleavage by Cas9 may be much more efficient than I-SceI [ 6 , 8 , 9 , 34 , 35 ]. Thus, in cells expressing abundant Cas9-sgRNA, these target sites could be efficiently recleaved and repaired until indels are introduced and accumulated (Fig.…”

“…While classical NHEJ (c-NHEJ) is the primary NHEJ pathway, alternative end joining (a-EJ) could also be employed to re-ligate the ends of DSBs if either of the core NHEJ factors including DNA-PKcs, Ku70/Ku80, and XRCC4/DNA ligase 4 is deficient or not engaged [4,5]. If the ends of DSBs are readily ligatable, such as Cas9-induced blunt ends and I-SceIinduced 3′-overhanging ends, c-NHEJ generates largely accurate end-joining products whereas a-EJ remains mostly mutagenic [6][7][8][9]. Additionally, using homologous sequences as a template, HDR is the preferred pathway for accurate substitutions and insertions in CRISPR/Cas9 genome editing.…”

Exonucleases: Degrading DNA to Deal with Genome Damage, Cell Death, Inflammation and Cancer

“…TREX2 facilitates nuclear DNA degradation in stressed keratinocytes, thus promoting cell death [ 22 , 224 , 226 , 227 ]. Interestingly, TREX2 has been shown to improve targeted CRISPR/Cas9 efficiency [ 228 , 229 , 230 ] by increasing deletion size and preventing perfect DNA repair, thereby avoiding repeated Cas9 cleavage and chromosomal translocations [ 231 ].…”

A Novel Anti-Cancer Therapy: CRISPR/Cas9 Gene Editing

“…The new types of Cas9, such as saCas9 and Cpf1, have been developed but cannot completely get rid of the dependence on PAM (Wang et al, 2018). This year, the newest types of Cas9-Cas9TX can inhibit the occurrence of chromosomal structural abnormalities such as chromosomal translocations and large fragment deletions in the process of gene editing and greatly improve the safety of CRISPR/ Cas9 gene editing (Yin et al, 2022).…”