Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems

By James E. DiCarlo, J. Norville, P. Mali, Xavier Rios, John D Aach, George M. Church. Nucleic Acids Research, 2013

1,548 citations recorded by the source corpus.

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.

Subfields: Genetics, Insect Science, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/0f7a4316af3da8877d56889b6468cccaf4ccf4f6

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Citations in the corpus

CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering (2013, background): “25μg of reporter library; or (3) 2μg control-TF plasmid and 0.”
Efficient Marker-Free Recovery of Custom Genetic Modifications with CRISPR/Cas9 in Caenorhabditis elegans (2014, background): “…activities that have recently revolutionized genome editing in a wide range of organisms (Wang et al. 1999; Chiu et al. 2013; Cho et al. 2013; Dicarlo et al. 2013; Friedland et al. 2013; Gratz et al. 2013; Hwang et al. 2013; Jiang et al. 2013; Katic and Großhans 2013; Li et al. 2013; Lo et…”
Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice (2013, neutral): “in bacteria (30,32), yeast (33), zebrafish (34–36), fruit fly (37) and human cells (28,29,38)].”
Highly Efficient Targeted Mutagenesis of Drosophila with the CRISPR/Cas9 System (2013, background): “…nucleases to selected target sites in the genome, making it possible to engineer mutations in a number of model organisms (Cong et al., 2013; DiCarlo et al., 2013; Gaj et al., 2013; Hwang et al., 2013; Mali et al., 2013), including flies (Beumer et al., 2008; Gratz et al., 2013; Liu et al.,…”
CRISPR interference (CRISPRi) for sequence-specific control of gene expression (2013, background): “Indeed, recent work has shown that this system can be used for efficient and multiplexed genome editing in a broad range of organisms including bacteria 19 , yeast 20 , fish 21 , mice 22 and human cells 23–26 .”