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Construction of a CRISPR/Cas9-Mediated Genome Editing System in Lentinula edodes.

ABSTRACT: CRISPR/Cas9 genome editing systems have been established in a broad range of eukaryotic species. Herein, we report the first method for genetic engineering in pyogo (shiitake) mushrooms (Lentinula edodes) using CRISPR/Cas9. For in vivo expression of guide RNAs (gRNAs) targeting the mating-type gene HD1 (LeA1), we identified an endogenous LeU6 promoter in the L. edodes genome. We constructed a plasmid containing the LeU6 and glyceraldehyde-3-phosphate dehydrogenase (LeGPD) promoters to express the Cas9 protein. Among the eight gRNAs we tested, three successfully disrupted the LeA1 locus. Although the CRISPR-Cas9-induced alleles did not affect mating with compatible monokaryotic strains, disruption of the transcription levels of the downstream genes of LeHD1 and LeHD2 was detected. Based on this result, we present the first report of a simple and powerful genetic manipulation tool using the CRISPR/Cas9 toolbox for the scientifically and industrially important edible mushroom, L. edodes.

SUBMITTER: Moon S

PROVIDER: S-EPMC8725921 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Construction of a CRISPR/Cas9-Mediated Genome Editing System in Lentinula edodes.

Moon Suyun S An Jee Young JY Choi Yeon-Jae YJ Oh Youn-Lee YL Ro Hyeon-Su HS Ryu Hojin H

Mycobiology 20211125 6

CRISPR/Cas9 genome editing systems have been established in a broad range of eukaryotic species. Herein, we report the first method for genetic engineering in pyogo (shiitake) mushrooms (Lentinula edodes) using CRISPR/Cas9. For in vivo expression of guide RNAs (gRNAs) targeting the mating-type gene HD1 (LeA1), we identified an endogenous LeU6 promoter in the L. edodes genome. We constructed a plasmid containing the LeU6 and glyceraldehyde-3-pho ...[more]

PMID: 35035251

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Project description:UnlabelledThe prokaryotic CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, an RNA-guided endonuclease, has been shown to mediate efficient genome editing in a wide variety of organisms. In the present study, the CRISPR-Cas9 system has been adapted to Leishmania donovani, a protozoan parasite that causes fatal human visceral leishmaniasis. We introduced the Cas9 nuclease into L. donovani and generated guide RNA (gRNA) expression vectors by using the L. donovani rRNA promoter and the hepatitis delta virus (HDV) ribozyme. It is demonstrated within that L. donovani mainly used homology-directed repair (HDR) and microhomology-mediated end joining (MMEJ) to repair the Cas9 nuclease-created double-strand DNA break (DSB). The nonhomologous end-joining (NHEJ) pathway appears to be absent in L. donovani. With this CRISPR-Cas9 system, it was possible to generate knockouts without selection by insertion of an oligonucleotide donor with stop codons and 25-nucleotide homology arms into the Cas9 cleavage site. Likewise, we disrupted and precisely tagged endogenous genes by inserting a bleomycin drug selection marker and GFP gene into the Cas9 cleavage site. With the use of Hammerhead and HDV ribozymes, a double-gRNA expression vector that further improved gene-targeting efficiency was developed, and it was used to make precise deletion of the 3-kb miltefosine transporter gene (LdMT). In addition, this study identified a novel single point mutation caused by CRISPR-Cas9 in LdMT (M381T) that led to miltefosine resistance, a concern for the only available oral antileishmanial drug. Together, these results demonstrate that the CRISPR-Cas9 system represents an effective genome engineering tool for L. donovani.ImportanceLeishmania donovani is the causative agent of fatal visceral leishmaniasis. To understand Leishmania infection and pathogenesis and identify new drug targets for control of leishmaniasis, more-efficient ways to manipulate this parasite genome are required. In this study, we have implemented CRISPR-Cas9 genome-editing technology in L. donovani. Both single- and dual-gRNA expression vectors were developed using a strong RNA polymerase I promoter and ribozymes. With this system, it was possible to generate loss-of-function insertion and deletion mutations and introduce drug selection markers and the GFP sequence precisely into the L. donovani genome. These methods greatly improved the ability to manipulate this parasite genome and will help pave the way for high-throughput functional analysis of Leishmania genes. This study further revealed that double-stranded DNA breaks created by CRISPR-Cas9 were repaired by the homology-directed repair (HDR) pathway and microhomology-mediated end joining (MMEJ) in Leishmania.

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Construction of a CRISPR/Cas9-Mediated Genome Editing System in Lentinula edodes.

Publications

Construction of a CRISPR/Cas9-Mediated Genome Editing System in <i>Lentinula edodes</i>.

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