Generation of dual-gRNA expressing vectors.

<p>(A) Schematic of dual-gRNA vectors. (B) Golden gate cloning strategy for insertion of specific guide sequences into each cassette. Note that the BbsI sites generate different overhangs after restriction digest. Red highlights indicate the BbsI sites, yellow and green highlights are part of hU6 promoter and gRNA, respectively, that are necessarily present in the plasmid. Blue and purple highlights indicate the unique customized guide sequences (C) One-step cloning protocol for the generation of customized dual-gRNA vectors. (D) Insertion of Sox1A and Sox3A oligonucleotide duplexes into pDG459 resulted in correct insertions in all 12 colonies as indicated by BspMI and SacI restriction digest. The black arrow indicates the diagnostic band for correct insertion.