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 has generally been considered a highly efficient and stochastic repair process. Yet recent studies indicated that c-NHEJ-mediated CRISPR-Cas9 mutagenesis, primarily leading to 1-bp insertions and small deletions, may be highly predictable. This observation has spurred the development of in-silico programs with remarkable accuracy in various animal species. Nevertheless, the predictability of CRISPR-induced mutation profiles across species and the associated mechanisms remained elusive. Through comparison of CRISPR-Cas9 repair outcomes between human and plant species, we discovered significant differences in 1-bp insertion profiles across species, governed by an X-family DNA polymerase, DNA Pol λ. The high predictability observed in human cells seemed linked to the template-dependent activity of human DNA Pol λ. However, this predictability cannot be directly extended to plants due to the dual activities of the plant homolog, capable of generating 1-bp insertions through both templated and non-templated manners. While knocking out DNA Pol λ led to deletion-only mutations, its overexpression enhanced 1-bp insertion by 1.2 - 2 folds at multiple target sites in plants. These findings unveiled the underlying mechanism dictating unique CRISPR-Cas9-induced insertion profiles across species and presented new strategies for achieving predictable and precise genome editing through c-NHEJ.