CRISPR-Cas12a induced DNA double-strand breaks are repaired by locus-dependent and error-prone pathways in a fungal pathogen

Abstract

CRISPR-Cas mediated genome engineering has revolutionized functional genomics. However, basic questions remain regarding the mechanisms of DNA repair following Cas-mediated DNA cleavage. We developed CRISPR-Cas12a ribonucleoprotein genome editing in the fungal plant pathogen, Magnaporthe oryzae, and found frequent donor DNA integration despite the absence of long sequence homology. Interestingly, genotyping from hundreds of transformants showed that frequent non-canonical DNA repair outcomes predominated the recovered genome edited strains. Detailed analysis using sanger and nanopore long-read sequencing revealed five classes of DNA repair mutations, including single donor DNA insertions, concatemer donor DNA insertions, large DNA deletions, deletions plus donor DNA insertions, and infrequently we observed INDELs. Our results show that different error-prone DNA repair pathways resolved the Cas12a-mediated double-strand breaks (DSBs) based on the DNA sequence of edited strains. Furthermore, we found that the frequency of the different DNA repair outcomes varied across the genome, with some tested loci resulting in more frequent large-scale mutations. These results suggest that DNA repair pathways provide preferential repair across the genome that could create biased genome variation, which has significant implications for genome engineering and the genome evolution in natural populations.

CRISPR-Cas12a ribonucleoprotein-mediated gene editing in the plant pathogenic fungus Magnaporthe oryzae

“…We amplified G418 coding sequence from plasmid pFGL921 and Bialaphos coding DNA from plasmid pBV9/pSM324 ( Kankanala et al., 2007 ; Zhang et al., 2021 ). If the gene being edited can be directly used for selection, such as editing FKBP12 ( Huang et al., 2021 ) or URA3 / URA5 as has been shown in Fusarium oxysporum for instance ( Wang et al., 2018 ), the inclusion of donor DNA in the experiment is not necessary. Note: Selection based on URA3 / URA5 has not been established in M. oryzae , and preliminary experiments might be needed to confirm selection based on URA3 / URA5 in M. oryzae .…”

“…Place the tube at room temperature for 20–25 min. Note: If your gene of interest can be used as selection directly, DNA donor is not required in step 22 For example, we have generated fkbp12 (FK506 receptor) mutants by using the selection of drug FK506 without DNA donor successfully ( Huang et al., 2021 ). Slowly add 1,000 μL PTC solution to the 50 mL falcon tube.…”

“…Note: The expected amplification size for wild-type is 1,648 bp, the size for mutants might vary based on the mutation pattern ( Huang et al., 2021 ).…”

“…We demonstrate this protocol using Magnaporthe oryzae (synonym of Pyricularia oryzae ), a model plant pathogenic fungus that is used to study plant-fungal interactions. For complete details on the use and execution of this protocol, please refer to Huang et al. (2021) .…”

“…For complete details on the use and execution of this protocol, please refer to Huang et al. (2021) .…”