Non-homologous end-joining-deficient filamentous fungal strains mitigate the impact of off-target mutations during the application of CRISPR/Cas9

Abstract

CRISPR/Cas9 genome editing technology has been implemented in almost all living organisms. Its editing precision appears to be very high and therefore could represent a big change from conventional genetic engineering approaches. However, guide RNA binding to nucleotides similar to the target site could result in undesired off-target mutations. Despite this, evaluating whether mutations occur is rarely performed in genome editing studies. In this study, we generated CRISPR/Cas9-derived filamentous fungal strains and analyzed them for the occurrence of mutations, and to which extent genome stability affects their occurrence. As a test case, we deleted the (hemi-)cellulolytic regulator-encoding gene xlnR in two Aspergillus niger strains: a wild type (WT) and a non-homologous end-joining (NHEJ)-deficient strain Δ kusA . Initial phenotypic analysis suggested a much higher prevalence of mutations in the WT compared to NHEJ-deficient strains, which was confirmed and quantified by whole-genome sequencing analysis. Our results clearly demonstrate that CRISPR/Cas9 applied to an NHEJ-deficient strain is an efficient strategy to avoid unwanted mutations. IMPORTANCE Filamentous fungi are commonly used biofactories for the production of industrially relevant proteins and metabolites. Often, fungal biofactories undergo genetic development (genetic engineering, genome editing, etc.) aimed at improving production yields. In this context, CRISPR/Cas9 has gained much attention as a genome editing strategy due to its simplicity, versatility, and precision. However, despite the high level of accuracy reported for CRISPR/Cas9, in some cases unintentional cleavages in non-targeted loci—known as off-target mutations—could arise. While biosafety should be a central feature of emerging biotechnologies to minimize unintended consequences, few studies quantitatively evaluate the risk of off-target mutations. This study demonstrates that the use of non-homologous end-joining-deficient fungal strains drastically reduces the number of unintended genomic mutations, ensuring that CRISPR/Cas9 can be safely applied for strain development.

Oligonucleotide-based CRISPR-Cas9 toolbox for efficient engineering ofKomagataella phaffii

“…Disruption of NHEJ pathway in K. phaffii and higher eukaryotes improves gene targeting and reduces unwanted genome-wide mutation in CRISPR-based experiments (Garrigues et al, 2023; Schusterbauer et al, 2022). However, the presence of the ku70 mutation may influence the final phenotype of the engineered strain.…”

“…DNA double strand breaks induced by a CRISPR nuclease allows easy gene inactivation via erroneous NHEJ based repair, as well as efficient template guided gene editing mediated by HR. However, recent studies have demonstrated that CRISPR nuclease induced DNA double strand breaks, despite the intended edits, also induce unwanted off-target mutations and/or result in gross chromosomal rearrangements in NHEJ-proficient strain backgrounds (Garrigues et al, 2023; Schusterbauer et al, 2022). Thus to minimize occurrence of unwanted mutations and improve gene targeting efficiency it is desirable to inactivate the NHEJ pathway.…”