Fig. 6.

Using CRISPR/Cas for genome editing. a Schematic showing how Cas9-gRNA generate DSBs at programmed target sites. b–f Different expression systems for CRISPR/Cas9 editing in yeast. b A yeast strain expressing inducible Cas9 from an integrated cassette requires a plasmid expressing sgRNA. c A CRISPR/Cas expression system requiring only one plasmid expressing both Cas9 and sgRNA. d A multiplex CRISPR/Cas system expressing Cas9, a tracrRNA, and a CRISPR/Cas array containing different crRNAs. e A multiplex CRISPR/Cas system expressing Cas9 and two or more sgRNAs on separate plasmids. f A multiplex CRISPR/Cas system expressing Cas9 from one plasmid, and using in vivo gap repair of a selectable plasmid by linear sgRNA cassettes to generate the sgRNA expression plasmid. g Schematic showing how nCas9 (D10A mutation in the RuvC1 endonuclease active site) generates a nick on the opposite strand of the target site. h Schematic showing how nCas9 (H840A mutation in the HNH endonuclease active site) generates a nick on the same strand of the target site. i Schematic showing how catalytically inactive dCas9 can be fused to an activation domain (AD) to recruit RNA polymerase (RNAP) and promote transcription of the target gene. j Schematic showing how catalytically inactive dCas9 can be fused to a repressor domain (RD) to inhibit RNA polymerase (RNAP) and prevent transcription of the target gene. k Schematic showing how catalytically inactive dCas9 can be fused to cytidine deaminase (CD) to convert a cytosine to a uracil. A round of repair and replication either converts the U back to C, or converts the U to a T to generate a TGA STOP codon. PAM: protospacer adjacent motif, gRNA: guide RNA including the Cas-interacting stem-loop structure, tracrRNA: gRNA portion that hybridizes with crRNA and contains stem-loop, crRNA: gRNA portion that hybridizes with crRNA and contains DNA targeting sequence, sgRNA: a gRNA that combines the tracrRNA and crRNA in a single RNA molecule, AD: activation domain, RD: repressor domain, CD: cytidine deaminase, RNAP: RNA polymerase II, TSS: transcription start site