Fig. 1.

Golden Gate Assembly of CRISPR gRNA expression array for simultaneously targeting of multiple genomic loci. Schematic illustration of Golden Gate Assembly of gRNA expression array using single modular, “array” vectors and the pMsgRNA-EGFP plasmid. Each gRNA guide oligonucleotide (T1 to T10, T11 to T20, or T21 to T30) is cloned into single modular plasmids (pMA1 to pMA10) based using the type IIS restriction enzyme BbsI correspondingly. The single pMA modular plasmid containing one gRNA expression cassette can be cut out and assembled into the “array” plasmids using the type IIS restriction enzyme BsaI, which will generate gRNA expression arrays carrying up to 10 cassettes. The gRNA expression arrays in the “array” plasmids can be further cut out and assembled into the pMsgRNA-EGFP plasmid using BsmBI, generating gRNA expression array containing 11–30 cassettes. GGC, Golden Gate Cloning; U6, U6 promoter for gRNA transcription; BB, BbsI restriction enzyme; Scr, SpCas9 gRNA scaffold sequences; EGFP, EGFP expression cassette driven by the CMV promoter