Title: Establishment of a genome editing tool using CRISPR-Cas9 ribonucleoprotein complexes in the non-model plant pathogen Sphaerulina musiva

Abstract

CRISPR-Cas9 is a versatile genome editing system widely used since 2013 to introduce site-specific modifications into the genomes of model and non-model species. This technology is used in various applications, from gene knock-outs, knock-ins, and over-expressions to more precise changes, such as the introduction of nucleotides at a targeted locus. CRISPR-Cas9 has been demonstrated to be easy to establish in new species and highly efficient and specific compared to previous gene editing strategies such as Zinc finger nucleases and transcription activator-like effector nucleases. Grand challenges for emerging CRISPR-Cas9 tools in filamentous fungi are developing efficient transformation methods for non-model organisms. In this paper, we have leveraged the establishment of CRISPR-Cas9 genome editing tool that relies on Cas9/sgRNA ribonucleoprotein complexes (RNPs) in the model species Trichoderma reesei and developed the first protocol to efficiently transform the non-model species, Sphaerulina musiva. This fungal pathogen constitutes a real threat to the genus Populus, a foundational bioenergy crop used for biofuel production. Herein, we highlight the general considerations to design sgRNAs and their computational validation. We also describe the use of isolated protoplasts to deliver the CRISPR-Cas9 RNP components in both species and the screening for targeted genome editing events. The development of engineeringmore » tools in S. musiva can be used for studying genes involved in diverse processes such as secondary metabolism, establishment, and pathogenicity, among many others, but also for developing genetic mitigation approaches. The approach described here provides guidance for potential development of transformation systems in other non-model spore-bearing ascomycetes.« less

Authors:

Tannous, Joanna; Sawyer, Cole; Hassan, Md Mahmudul; Labbe, Jesse L.; Eckert, Carrie

Publication Date:

Fri Jul 21 00:00:00 EDT 2023

Research Org.:

Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER)

OSTI Identifier:

1992008

Alternate Identifier(s):

OSTI ID: 1992743

Grant/Contract Number:  

AC05-00OR22725

Resource Type:

Published Article

Journal Name:

Frontiers in Genome Editing

Additional Journal Information:

Journal Name: Frontiers in Genome Editing Journal Volume: 5; Journal ID: ISSN 2673-3439

Publisher:

Frontiers Media SA

Country of Publication:

Switzerland

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; CRISPR-Cas9; RNP; spore-bearing fungi; ascomycetes; trichoderma reesei; Sphaerulina musiva