Dual activities of an X-family DNA polymerase regulate CRISPR-induced insertional mutagenesis across species

Abstract

The canonical non-homologous end joining (c-NHEJ) repair pathway, generally viewed as stochastic, has recently been shown to produce predictable outcomes in CRISPR-Cas9 mutagenesis. This predictability, mainly in 1-bp insertions and small deletions, has led to the development of in-silico prediction programs for various animal species. However, the predictability of CRISPR-induced mutation profiles across species remained elusive. Comparing CRISPR-Cas9 repair outcomes between human and plant species reveals significant differences in 1-bp insertion profiles. The high predictability observed in human cells links to the template-dependent activity of human Polλ. Yet plant Polλ exhibits dual activities, generating 1-bp insertions through both templated and non-templated manners. Polλ knockout in plants leads to deletion-only mutations, while its overexpression enhances 1-bp insertion rates. Two conserved motifs are identified to modulate plant Polλ‘s dual activities. These findings unveil the mechanism behind species-specific CRISPR-Cas9-induced insertion profiles and offer strategies for predictable, precise genome editing through c-NHEJ.

Context effects on repair of 5’-overhang DSB induced by Cas12a in Arabidopsis

“…The majority of studies DSB-induced DNA repair/recombination in plants in recent years have involved the Cas9 endonuclease and its derivatives. Sequentially cleaving the two strands of the target DNA, Cas9 generates a blunt-ended or 1-base overhang DSB (Jinek et al, 2012;Kumar et al, 2023;Longo et al, 2024;Swarts and Jinek, 2018;Weiss et al, 2024). This means that most studies of Cas9-induced breaks involve the study of blunt-ended DSB, without single-stranded (ssDNA) overhangs.…”