Background

The sgRNA of the CRISPR-Cas9 system is mainly promoted by the small nuclear RNA promoters such as U6 and U3. Although it has been tested with prospered efficiency in many cases, it also 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. The traditional RNA polymerase II promoter can’t be used in driving sgRNA expression, extra nucleotides will be added to the 5’- and 3’-ends of gRNA by RNA polymerase II and may interrupt the normal gRNA function. Additionally, RNAs transcribed by RNA polymerase II are exported rapidly into the cytoplasm while nuclear localization is required for the CRISPR-Cas9/gRNA duplex to access the genome editing (Lei et al., 2001). To overcome these obstacles, we use the ribozyme’s self-catalyzed cleavage to release the precise processing mature sgRNA under a RNA polymerase II promoter which drives expression of both Cas9 and sgRNA (named STU CRISPR-Cas9 system, Figure 1). Compared to the traditional small nuclear RNA promoters used in sgRNA expression, our STU CRISPR-Cas9 system has some advantages: (1) it’s shorter and easier in vector construction, and it will increase the transformation efficiency under some circumstances; (2) it only needs extra ribozyme flanking sequence (shorter than any RNA polymerase III promoter we are currently using) for multiple sgRNAs expression;(3) it has shown higher deletion efficiency induced by double sgRNAs. Thus, the STU CRISPR-Cas9 system driven by a single RNA polymerase II promoter can replace the traditional CRISPR-Cas9 system now we are using whether in vivo or in vitro if appropriate promoters are chosen.


Figure 1. Schematic illustration of the single transcription unit (STU) CRISPR-Cas9 system. Once transcribed by a Pol II promoter, the STU CRISPR-Cas9 primary transcripts will undergo self-cleavage by hammerhead ribozyme (RZ) to release the mature Cas9 mRNA and sgRNA. The Cas9 mRNA is terminated with a synthetic polyA (pA) sequence to facilitate translation, The RZ sequence (in blue) and its target sequence (in black) are illustrated.