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

Abstract

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, ∼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 ∼4.2 kb of coding sequence, as well as the development of single guide RNA (sgRNA) expression cassettes substantially smaller than the current ∼360 bp size. Here, we report that small, ∼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.

CRISPR/Cas9 with single guide RNA expression driven by small tRNA promoters showed reduced editing efficiency compared to a U6 promoter

“…This result is in contrast to the conclusions reported by Mefferd et al (2015), the robust production of sgRNAs driven by tRNA promoters as well as comparable editing efficiency between CRISPR/Cas9 with tRNA promoter-and U6 promoter-expressed sgRNAs. We note two major differences between our experimental systems that might be able to explain the contradictory results.…”

“…(ii) Different targeting sequences were used. Mefferd et al (2015) noted in their study that targeting sequences with high GC content could inhibit tRNase Z processing, suggesting that expression efficiency of sgRNAs driven by tRNA promoters could be sequence-dependent. Among the sgRNA sequences tested in our systems, one is GC rich (Pcsk9, GC content = 71%), with the others having normal GC content (Apoc3, GC content = 59%; Mkk4, GC content = 38%, Mstn, GC content = 55%; PCSK9, GC content = 65%).…”

“…As AAV vectors have a DNA packaging limit of ∼5 kb, and the standard AAV-SaCRISPR system almost fills the cargo limitation, it would be helpful to further reduce the total length of the SaCRISPR system in order to make extra space for more sgRNA cassettes and to improve the packaging efficiency. Mefferd et al (2015) reported recently that expression of sgRNAs can be driven by small tRNA promoters (∼70 bp), making it possible to express two full-length sgRNAs using only ∼350 bp of space, about the size of one sgRNA-expressing cassette with a U6 promoter. We tested the potential of this approach by first comparing side-by-side the genome editing efficiency of CRISPR/Cas9 with U6 promoter-and tRNA promoter-driven sgRNA expression, respectively.…”

“…For efficient delivery of CRISPR materials to cells in vitro, we reconstructed the original lentiCRISPR v2 for Streptococcus pyogenes (SpCas9) targeting (Sanjana et al 2014) constructs with different sgRNA promoters-U6pro, tRNA GLN pro, and tRNA PRO pro-with the latter two tRNA promoters reported to be able to effectively drive the expression of sgRNAs in SaCRISPR mediated targeting ( Fig. 1A; Mefferd et al 2015). We next made constructs with different lentiSaCRISPR vectors targeting several mouse genes as well as a human gene, using previously screened sgRNA sequences that had shown high on-target efficiency with a U6 promoter.…”

“…The gRNA sequences are listed in Supplemental Table 1. sgRNA expression driven by tRNA promoters www.rnajournal.org 3 to study CRISPR activities. In the Mefferd et al (2015) study, genome-editing activities with different promoter-driven sgRNAs were only studied with exogenous cotransfected indicator plasmids instead of directly assessing the endogenous genomic loci. As is shown in our study, while significant editing activity (>50%) was detected with indicator plasmids, editing activity was barely notable with Surveyor assays when targeting the endogenous genomic locus.…”