CRISPR-Cas12a induced DNA double-strand breaks are repaired by multiple pathways with different mutation profiles in Magnaporthe oryzae

Abstract

CRISPR-Cas mediated genome engineering has revolutionized functional genomics. However, understanding of DNA repair following Cas-mediated DNA cleavage remains incomplete. Using Cas12a ribonucleoprotein genome editing in the fungal pathogen, Magnaporthe oryzae, we detail non-canonical DNA repair outcomes from hundreds of transformants. Sanger and nanopore sequencing analysis reveals significant variation in DNA repair profiles, ranging from small INDELs to kilobase size deletions and insertions. Furthermore, we find the frequency of DNA repair outcomes varies between loci. The results are not specific to the Cas-nuclease or selection procedure. Through Ku80 deletion analysis, a key protein required for canonical non-homologous end joining, we demonstrate activity of an alternative end joining mechanism that creates larger DNA deletions, and uses longer microhomology compared to C-NHEJ. Together, our results suggest preferential DNA repair pathway activity in the genome that can create different mutation profiles following repair, which could create biased genome variation and impact genome engineering and genome evolution.

CRISPR/dCas9-Mediated Gene Silencing in Two Plant Fungal Pathogens

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The Devastating Rice Blast Airborne Pathogen Magnaporthe oryzae—A Review on Genes Studied with Mutant Analysis

“…Among them, gene deletion via homologous recombination has been the most commonly used method in the past few decades. The latest CRISPR technology has also been developed to improve gene-editing efficiency [ 37 , 38 ]. The rest of this review will give an overview of all M. oryzae genes studied to date using such approaches ( Supplementary Table S1 ).…”

Advances and Challenges in CRISPR/Cas-Based Fungal Genome Engineering for Secondary Metabolite Production: A Review

“…The results suggest that the gene editing efficiency is affected by the Cas nuclease and the target locus. A very recent study using sanger and nanopore sequencing analysis demonstrated that Cas12a-based ribonucleoprotein (RNP) could induce a spectrum of DNA mutations, ranging from small INDELs to large deletions and insertions, in the genome of M. oryzae , which suggests the involvement of multiple DNA-repair pathways in repairing the double-strand staggered break caused by Cas12a [ 74 , 75 ]. Interestingly, the biased DNA variations observed in this study suggested a hierarchy for DNA repair pathway choice, which might be mediated by the epigenome and has significant implications for genome engineering and evolution [ 24 , 74 ].…”