Advances in CRISPR Technologies for Microbial Strain Engineering

Alper, Hal S.; Beisel, Chase L.

doi:10.1002/biot.201800460

Cited by 2 publications

(1 citation statement)

References 16 publications

(16 reference statements)

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“…In particular, they have enabled rapid, easy yet accurate construction of strains for some production platform microorganisms, e.g. Saccharomyces cerevisiae and Escherichia coli (15)(16)(17)(18), contributing significantly to the bio-based economy. For the bio-based economy, other alternative production microorganisms are also essentially required to generate sustainable alternatives for yielding green chemicals and biofuels from environmentally friendly resources, and become increasingly important.…”

Section: Introductionmentioning

confidence: 99%

Characterization and repurposing of the endogenous Type I-F CRISPR-Cas system ofZymomonas mobilisfor genome engineering

Zheng

Han

Liang

et al. 2019

Preprint

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Establishment of production platform organisms through prokaryotic engineering represents an efficient means to generate alternatives for yielding renewable biochemicals and biofuels from sustainable resources. Zymomonas mobilis, a natural facultative anaerobic ethanologen, possesses many attractive physiological attributes, making it an important industrial microorganism. To facilitate the broad applications of this strain for biorefinery, an efficient genome engineering toolkit for Z.mobilis was established in this study by repurposing the endogenous Type I-F CRISPR-Cas system upon its functional characterization, and further updated. This toolkit includes a series of genome engineering plasmids, each carrying an artificial self-targeting CRISPR and a donor DNA for the recovery of recombinants. Using the updated toolkit, genome engineering purposes were achieved with efficiencies of up to 100%, including knockout of cas3 gene, replacement of cas3 with the mCherry-encoding rfp gene, nucleotide substitutions in cas3, and deletion of two large genomic fragments up to 10 kb. This study established thus far the most efficient, straightforward and convenient genome engineering toolkit for Z. mobilis, and laid a foundation for further native CRISPRi studies in Z. mobilis, which extended the application scope of CRISPR-based technologies, and could also be applied to other industrial microorganisms with unexploited endogenous CRISPR-Cas systems.

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Section: Introductionmentioning

confidence: 99%