A Single Transcript CRISPR-Cas9 System for Multiplex Genome Editing in Plants.

The CRISPR-Cas9 system has been widely adopted in genome editing. By changing the 20 bp guide sequence, it can easily edit any sequence adjacent to a protospacer adjacent motif (PAM) in a genome. Multiplex genome editing could be accomplished with simultaneous expression of multiple single-guide RNAs (sgRNA). Given that sgRNAs are expressed by Pol III promoters, multiplex genome editing is conventionally done by assembly of multiple complete sgRNA expression cassettes together, which can be a challenge in vector construction. Here, we described a multiplex genome editing system based on a single transcript unit CRISPR-Cas9 (STU CRISPR-Cas9) expression system driven by a single Pol II promoter. It represents a novel approach for multiplex genome editing.