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Multiplexed Guide R...
Multiplexed Guide RNA Expression Leads to Increased Mutation Frequency in Targeted Window Using a CRISPR-Guided Error-Prone DNA Polymerase in Saccharomyces cerevisiae
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- Gossing, Michael (author)
- AstraZeneca AB
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- Limeta, Angelo, 1996 (author)
- Chalmers tekniska högskola,Chalmers University of Technology
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- Skrekas, Christos, 1990 (author)
- Chalmers tekniska högskola,Chalmers University of Technology
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- Wigglesworth, Mark (author)
- AstraZeneca AB
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- Davis, Andrew (author)
- AstraZeneca AB
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- Siewers, Verena, 1976 (author)
- Chalmers tekniska högskola,Chalmers University of Technology
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- David, Florian, 1981 (author)
- Chalmers tekniska högskola,Chalmers University of Technology
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(creator_code:org_t)
- 2023
- 2023
- English.
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In: ACS Synthetic Biology. - 2161-5063. ; 12:8, s. 2271-2277
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https://research.cha...
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https://doi.org/10.1...
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Abstract
Subject headings
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- Clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9technology, with its ability to target a specific DNA locus usingguide RNAs (gRNAs), is particularly suited for targeted mutagenesis.The targeted diversification of nucleotides in Saccharomycescerevisiae using a CRISPR-guided error-prone DNA polymerase calledyEvolvR was recently reported. Here, we investigate the effectof multiplexed expression of gRNAs flanking a short stretch of DNAon reversion and mutation frequencies using yEvolvR. Phenotypic assaysdemonstrate that higher reversion frequencies are observed when expressingmultiple gRNAs simultaneously. Next generation sequencing revealsa synergistic effect of multiple gRNAs on mutation frequencies, whichis more pronounced in a mutant with a partially defective DNA mismatchrepair system. Additionally, we characterize a galactose-inducibleyEvolvR, which enables temporal control of mutagenesis. This studydemonstrates that multiplex expression of gRNAs and induction of mutagenesisgreatly improves the capabilities of yEvolvR for generation of geneticlibraries in vivo.
Subject headings
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
- MEDICIN OCH HÄLSOVETENSKAP -- Medicinska och farmaceutiska grundvetenskaper -- Cell- och molekylärbiologi (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Basic Medicine -- Cell and Molecular Biology (hsv//eng)
Keyword
- CRISPR
- directed evolution
- multiplexing
- in vivo mutagenesis
- targeted mutagenesis
- yeast
Publication and Content Type
- art (subject category)
- ref (subject category)
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Gossing, Michael
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Limeta, Angelo, ...
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Skrekas, Christo ...
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Wigglesworth, Ma ...
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Davis, Andrew
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Siewers, Verena, ...
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show more...
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David, Florian, ...
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show less...
- About the subject
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- NATURAL SCIENCES
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NATURAL SCIENCES
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and Biological Scien ...
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and Biochemistry and ...
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- MEDICAL AND HEALTH SCIENCES
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MEDICAL AND HEAL ...
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and Basic Medicine
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and Cell and Molecul ...
- Articles in the publication
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ACS Synthetic Bi ...
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Chalmers University of Technology