Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems
Open accessAbstract
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems in bacteria and archaea use RNA-guided nuclease activity to provide adaptive immunity against invading foreign nucleic acids. Here, we report the use of type II bacterial CRISPR-Cas system in Saccharomyces cerevisiae for genome engineering. The CRISPR-Cas components, Cas9 gene and a designer genome targeting CRISPR guide RNA (gRNA), show robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci in yeast. Using constitutive Cas9 expression and a transient gRNA cassette, we show that targeted double-strand breaks can increase homologous recombination rates of single- and double-stranded oligonucleotide donors by 5-fold and 130-fold, respectively. In addition, co-transformation of a gRNA plasmid and a donor DNA in cells constitutively expressing Cas9 resulted in near 100% donor DNA recombination frequency. Our approach provides foundations for a simple and powerful genome engineering tool for site-specific mutagenesis and allelic replacement in yeast.
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The type II CRISPR/Cas9 system has been exploited for high-efficiency gene editing in model microorganisms such as E. coli, S. cerevisiae, and Streptococcus pneumoniae.
“The type II CRISPR/Cas9 system has been exploited for high-efficiency gene editing in model microorganisms such as E. coli (Jiang et al., 2015), S. cerevisiae (DiCarlo et al., 2013), and Streptococcus pneumoniae (Jiang et al., 2013).”
From Gas Fermentation—A Flexible Platform for Commercial Scale Production of Low-Carbon-Fuels and Chemicals from Waste and Renewable Feedstocks (2016) · cites this paper as DiCarlo et al., 2013
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- Authors
- DiCarlo, J. E., Norville, J. E., Mali, P., Rios, X., Aach, J., & Church, G. M.
- Affiliations
- Boston University (US)
- Corresponding author
- Church, George M.
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2013-03-04
- Volume
- 41
- Issue
- 7
- Pages
- 4336-4343
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- PMC
- PMC3627607
- OpenAlex
- W2115420193
- Cited by
- 1,566
- References
- 23
- Citations by year
- 2026: 45 · 2025: 78 · 2024: 88 · 2023: 93 · 2022: 131 · 2021: 131 · 2020: 125 · 2019: 148 · 2018: 163 · 2017: 146 · 2016: 147 · 2015: 103 · 2014: 102 · 2013: 64
- FWCI
- 61.75
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Nucleic Acids Research (publishedVersion, cc-by-nc)
- Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
- PubMed Central (publishedVersion, cc-by-nc)
- PubMed (publishedVersion)
- OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion, other-oa)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
- Funders
- National Science Foundation
- U.S. Department of Energy (DEFG0202ER63445)
- National Institutes of Health
- National Human Genome Research Institute (P50HG005550)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Keywords
- CRISPR/Cas9, Saccharomyces cerevisiae, genome editing, RNA-guided endonuclease, homologous recombination, site-directed mutagenesis, allelic replacement, DNA double-strand breaks, co-transformation
- MeSH terms
- Endodeoxyribonucleases, Genes, Bacterial, Genetic Engineering, Plasmids, Saccharomyces cerevisiae, Polymerase Chain Reaction, Mutagenesis, Genome, Fungal, Inverted Repeat Sequences, Genetic Loci, RNA, Small Untranslated, Homologous Recombination
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