Construction of a Single Transcriptional Unit for Expression of Cas9 and Single-guide RNAs for Genome Editing in Plants.

The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein9 (Cas9) is a simple and efficient tool for genome editing in many organisms including plant and crop species. The sgRNAs of the CRISPR/Cas9 system are typically expressed from RNA polymerase III promoters, such as U6 and U3. In many transformation events, more nucleotides will increase the difficulties in plasmid construction and the risk of wrong integration in genome such as base-pair or fragment missing ( Gheysen et al., 1990 ). And also, in many organisms, Pol III promoters have not been well characterized, and heterologous Pol III promoters often perform poorly ( Sun et al., 2015 ). Thus, we have developed a method using single transcriptional unit (STU) CRISPR-Cas9 system to drive the expression of both Cas9 and sgRNAs from a single RNA polymerase II promoter to achieve effective genome editing in plants.