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Doxycyline regulation in a single retroviral vector by an autoregulatory loop facilitates controlled gene expression in liver cells

Nucleic Acids Research
Oxford University Press
Publication Date
DOI: 10.1093/nar/gnh034
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OP-NARE130135 4336..4343 Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems James E. DiCarlo1,2, Julie E. Norville2, Prashant Mali2, Xavier Rios2, John Aach2 and George M. Church2,* 1Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA and 2Department of Genetics, Harvard Medical School, Boston, MA 02115, USA Received January 10, 2013; Revised February 10, 2013; Accepted February 11, 2013 ABSTRACT 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 en- gineering. The CRISPR-Cas components, Cas9 gene and a designer genome targeting CRISPR guide RNA (gRNA), show robust and specific RNA-guided endo- nuclease 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 homolo- gous 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 con- stitutively 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 mutagen- esis and allelic replacement in yeast. INTRODUCTION Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) immune systems in bacteria are of interest to the biotech- nology community owing to RNA-guided endonuclease activity (1,2). The Cas9 gene, from the type II bacterial CRISPR system of Streptococcus pyogenes, complexes with a designer genome targeting CRISPR guide RNA (gRNA) to determine the site specificity of the DNA cutting activity (2,3) (Figure

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