title: mCAL: A New Approach for Versatile Multiplex Action of Cas9 Using One sgRNA and Loci Flanked by a Programmed Target Sequence.
abstract: Genome editing exploiting CRISPR/Cas9 has been adopted widely in academia and in the biotechnology industry to manipulate DNA sequences in diverse organisms. Molecular engineering of Cas9 itself and its guide RNA, and the strategies for using them, have increased efficiency, optimized specificity, reduced inappropriate off-target effects, and introduced modifications for performing other functions (transcriptional regulation, high-resolution imaging, protein recruitment, and high-throughput screening). Moreover, Cas9 has the ability to multiplex, i.e., to act at different genomic targets within the same nucleus. Currently, however, introducing concurrent changes at multiple loci involves: (i) identification of appropriate genomic sites, especially the availability of suitable PAM sequences; (ii) the design, construction, and expression of multiple sgRNA directed against those sites; (iii) potential difficulties in altering essential genes; and (iv) lingering concerns about "off-target" effects. We have devised a new approach that circumvents these drawbacks, as we demonstrate here using the yeast Saccharomyces cerevisiae First, any gene(s) of interest are flanked upstream and downstream with a single unique target sequence that does not normally exist in the genome. Thereafter, expression of one sgRNA and cotransformation with appropriate PCR fragments permits concomitant Cas9-mediated alteration of multiple genes (both essential and nonessential). The system we developed also allows for maintenance of the integrated, inducible Cas9-expression cassette or its simultaneous scarless excision. Our scheme-dubbed mCAL for " M: ultiplexing of C: as9 at A: rtificial L: oci"-can be applied to any organism in which the CRISPR/Cas9 methodology is currently being utilized. In principle, it can be applied to install synthetic sequences into the genome, to generate genomic libraries, and to program strains or cell lines so that they can be conveniently (and repeatedly) manipulated at multiple loci with extremely high efficiency.
authors.affiliation: Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202.
authors.forename: Gregory C
authors.initials: GC
authors.lastname: Finnigan
authors.affiliation: Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202 jthorner@berkeley.edu.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Thorner
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods*; D015870:Gene Expression; D005818:Genetic Engineering; D056426:Genetic Loci; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D029701:Saccharomyces cerevisiae Proteins; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
keywords: CRISPR; Saccharomyces cerevisiae; essential genes; genome editing; genome engineering