Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering
Abstract:BackgroundOver the past 3 years, the CRISPR/Cas9 system has revolutionized the field of genome engineering. However, its application has not yet been validated in thermophilic fungi. Myceliophthora thermophila, an important thermophilic biomass-degrading fungus, has attracted industrial interest for the production of efficient thermostable enzymes. Genetic manipulation of Myceliophthora is crucial for metabolic engineering and to unravel the mechanism of lignocellulose deconstruction. The lack of a powerful, v… Show more
“…Combining multiple sgRNAs in an array is particularly useful for R. toruloides, as this minimizes the amount of genetic material that needs to be delivered while maximizing the number of potential gene targets. Additionally, utilizing multiple sgRNAs to target one gene at different locations allows for the possibility of removing a large DNA in between the target sites, and also increases the possibility that the target gene is successfully disrupted (20, 46, 47). Therefore, our constructs were designed to express four sgRNAs (two for each gene) such that cleavage would occur at two sites separated by ∼500bp in both genes.…”
Section: Resultsmentioning
confidence: 99%
“…However, the process of delivering and expressing fully functional CRISPR components, and the biology of each organism’s DNA repair pathways, requires organism-specific optimization to accommodate each species’ unique characteristics. The past two years have seen the publication of a wide array of studies demonstrating CRISPR-Cas9 genome engineering in fungal species for which few robust DNA editing tools existed, including Aspergillus niger (49), Cryptococcus neoformans (50), Mucor circinelloides (51), and Myceliophthora thermophila (47). The dawning of a “fungal CRISPR revolution” has occurred, empowering researchers to explore new bioproduction possibilities in obscure yet promising fungi.…”
“…Combining multiple sgRNAs in an array is particularly useful for R. toruloides, as this minimizes the amount of genetic material that needs to be delivered while maximizing the number of potential gene targets. Additionally, utilizing multiple sgRNAs to target one gene at different locations allows for the possibility of removing a large DNA in between the target sites, and also increases the possibility that the target gene is successfully disrupted (20, 46, 47). Therefore, our constructs were designed to express four sgRNAs (two for each gene) such that cleavage would occur at two sites separated by ∼500bp in both genes.…”
Section: Resultsmentioning
confidence: 99%
“…However, the process of delivering and expressing fully functional CRISPR components, and the biology of each organism’s DNA repair pathways, requires organism-specific optimization to accommodate each species’ unique characteristics. The past two years have seen the publication of a wide array of studies demonstrating CRISPR-Cas9 genome engineering in fungal species for which few robust DNA editing tools existed, including Aspergillus niger (49), Cryptococcus neoformans (50), Mucor circinelloides (51), and Myceliophthora thermophila (47). The dawning of a “fungal CRISPR revolution” has occurred, empowering researchers to explore new bioproduction possibilities in obscure yet promising fungi.…”
“…7.4b) will enable faster construction of yieldoptimized strains and will fuel its use for rational design in strain construction as well as for more genome-wide searches for gene modifications that improve production of an enzyme. Genome editing in the thermophilic fungus Myceliophthora was used to increase cellulase production by 13-fold by editing up to four genes at one time (Liu et al 2017).…”
Section: Genome Editing As a Revolutionizing Tool For Production Stramentioning
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.