Dear Editor,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).To overcome these limitations,we sought to drive the expression of both Cas9 and sgRNAs from a single Pol Ⅱ promoter (either inducible or constitutive) to achieve effective genome editing.This would allow a better spatiotemporal control of these gene targeting reagents,and would be applicable tothe organisms where Pol Ⅲ promoters are not well characterized.