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

The Streptococcus pyogenes CRISPR-Cas9 system effectively mediates RNA-guided DNA double-strand breaks and is used for genome editing in many different organisms, including plants (Puchta, 2016). CRISPR-Cas9 is a two-component system in which the Cas9 protein is expressed from a Pol II promoter (Lowder et al., 2015). In contrast, the sgRNAs are typically expressed from Pol III promoters, such as U6 and U3. Although the CRISPR-Cas9 system has been proven very powerful for genome editing, it has some limitations: (1) it is hard to achieve coordinated and/or inducible expression of Cas9 and the sgRNAs; (2) manipulating multiple sgRNAs for multiplexed gene editing can be tedious, requiring multiple Pol III promoters; (3) in many non-model organisms, Pol III promoters have not been well characterized, and heterologous Pol III promoters often perform poorly (Sun et al., 2015).