Expression of CRISPR/Cas single guide RNAs using small tRNA promoters

The in vivo application of CRISPR/Cas-based DNA editing technology will require the development of efficient delivery methods that likely will be dependent on adeno-associated virus (AAV)-based viral vectors. However, AAV vectors have only a modest, similar to 4.7-kb packaging capacity, which will necessitate the identification and characterization of highly active Cas9 proteins that are substantially smaller than the prototypic Streptococcus pyogenes Cas9 protein, which covers similar to 4.2 kb of coding sequence, as well as the development of single guide RNA (sgRNA) expression cassettes substantially smaller than the current similar to 360 bp size. Here, we report that small, similar to 70-bp tRNA promoters can be used to express high levels of tRNA: sgRNA fusion transcripts that are efficiently and precisely cleaved by endogenous tRNase Z to release fully functional sgRNAs. Importantly, cells stably expressing functional tRNA: sgRNA precursors did not show a detectable change in the level of endogenous tRNA expression. This novel sgRNA expression strategy should greatly facilitate the construction of effective AAV-based Cas9/sgRNA vectors for future in vivo use.