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

Jun Huang, David Rowe, Pratima Subedi, Wei Zhang, Tyler Suelter, Barbara Valent and David E. Cook ()
Additional contact information
Jun Huang: Kansas State University
David Rowe: Kansas State University
Pratima Subedi: Kansas State University
Wei Zhang: Kansas State University
Tyler Suelter: Kansas State University
Barbara Valent: Kansas State University
David E. Cook: Kansas State University

Nature Communications, 2022, vol. 13, issue 1, 1-18

Abstract: 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.

Date: 2022
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DOI: 10.1038/s41467-022-34736-1

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