Dataset Information

The Cas9-gRNA ribonucleoprotein complex-mediated editing of pyrG in Ganoderma lucidum and unexpected insertion of contaminated DNA fragments.

ABSTRACT: Gene editing is a promising alternative to traditional breeding for the generation of new mushroom strains. However, the current approach frequently uses Cas9-plasmid DNA to facilitate mushroom gene editing, which can leave residual foreign DNA in the chromosomal DNA raising concerns regarding genetically modified organisms. In this study, we successfully edited pyrG of Ganoderma lucidum using a preassembled Cas9-gRNA ribonucleoprotein complex, which primarily induced a double-strand break (DSB) at the fourth position prior to the protospacer adjacent motif. Of the 66 edited transformants, 42 had deletions ranging from a single base to large deletions of up to 796 bp, with 30 being a single base deletion. Interestingly, the remaining 24 contained inserted sequences with variable sizes at the DSB site that originated from the fragmented host mitochondrial DNA, E. coli chromosomal DNA, and the Cas9 expression vector DNA. The latter two were thought to be contaminated DNAs that were not removed during the purification process of the Cas9 protein. Despite this unexpected finding, the study demonstrated that editing G. lucidum genes using the Cas9-gRNA complex is achievable with comparable efficiency to the plasmid-mediated editing system.

SUBMITTER: Eom H

PROVIDER: S-EPMC10333205 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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The Cas9-gRNA ribonucleoprotein complex-mediated editing of pyrG in Ganoderma lucidum and unexpected insertion of contaminated DNA fragments.

Eom Hyerang H Choi Yeon-Jae YJ Nandre Rutuja R Han Hui-Gang HG Kim Sinil S Kim Minseek M Oh Youn-Lee YL Nakazawa Takehito T Honda Yoichi Y Ro Hyeon-Su HS

Scientific reports 20230710 1

Gene editing is a promising alternative to traditional breeding for the generation of new mushroom strains. However, the current approach frequently uses Cas9-plasmid DNA to facilitate mushroom gene editing, which can leave residual foreign DNA in the chromosomal DNA raising concerns regarding genetically modified organisms. In this study, we successfully edited pyrG of Ganoderma lucidum using a preassembled Cas9-gRNA ribonucleoprotein complex, which primarily induced a double-strand break (DSB) ...[more]

PMID: 37429890

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Project description:Targeted gene insertion or replacement is a promising genome-editing tool for molecular breeding and gene engineering. Although CRISPR/Cas9 works well for gene disruption and deletion in Ganoderma lucidum, targeted gene insertion and replacement remain a serious challenge due to the low efficiency of homologous recombination (HR) in this species. In this work, we demonstrate that the DNA double-strand breaks induced by Cas9 were mainly repaired via the nonhomologous end joining (NHEJ) pathway, at a frequency of 96.7%. To establish an efficient target gene insertion and replacement tool in Ganoderma, we first inactivated the NHEJ pathway via disruption of the Ku70 gene (ku70) using a dual single guide RNA (sgRNA)-directed gene deletion method. Disruption of the ku70 gene significantly decreased NHEJ activity in G. lucidum. Moreover, ku70 disruption strains exhibited 96.3% and 93.1% frequencies of targeted gene insertion and replacement, respectively, when target DNA with the orotidine 5'-monophosphate decarboxylase (ura3) gene and 1.5-kb homologous 5'- and 3'-flanking sequences was used as a donor template, compared to 3.3% and 0%, respectively, at these targeted sites for a control strain (Cas9 strain). Our results indicated that ku70 disruption strains were efficient recipients for targeted gene insertion and replacement. This tool will advance our understanding of functional genomics in G. lucidum. IMPORTANCE Functional genomic studies in Ganoderma have been hindered by the absence of adequate genome-engineering tools. Although CRISPR/Cas9 works well for gene disruption and deletion in G. lucidum, targeted gene insertion and replacement have remained a serious challenge due to the low efficiency of HR in these species, although such precise genome modifications, including site mutations, site-specific integrations, and allele or promoter replacements, would be incredibly valuable. In this work, we inactivated the NHEJ repair mechanism in G. lucidum by disrupting the ku70 gene using the CRISPR/Cas9 system. Moreover, we established a target gene insertion and replacement method in ku70-disrupted G. lucidum that possessed high-efficiency gene targeting. This technology will advance our understanding of the functional genomics of G. lucidum.

Dataset Information

The Cas9-gRNA ribonucleoprotein complex-mediated editing of pyrG in Ganoderma lucidum and unexpected insertion of contaminated DNA fragments.

Publications

The Cas9-gRNA ribonucleoprotein complex-mediated editing of pyrG in Ganoderma lucidum and unexpected insertion of contaminated DNA fragments.

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