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Lewis Fry, Caroline Peddle, Alun R Barnard, Michelle E McClements, Robert E MacLaren; Guide RNA orientation influences Cas9 expression in a single vector approach to CRISPR editing in a model system. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4937. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Dominantly inherited mutations in rod specific genes including rhodopsin are a significant cause of retinitis pigmentosa, but are not amenable to treatment with simple gene replacement therapy. Approaches using AAV-delivered CRISPR-Cas9 to disrupt these pathogenic mutations have shown potential. Ideally this would be delivered using a single AAV vector for safe and efficient photoreceptor transduction. Although small Cas9 orthologues such as Staphyloccous Aureus Cas9 (SaCas9, 3.1kb) allow delivery of the Cas9 and gRNA within a single AAV, the transcriptional elements must be tightly arranged. To reduce transcriptional interference, we placed the gRNA scaffold element on the reverse strand (trans) from Cas9. We investigated whether this affected Cas9 and gRNA expression and knockdown of the EGFP gene in an in vitro model system.
Plasmids were generated to contain a CMV-IE promoter driven SaCas9, with a U6-driven gRNA scaffold placed on either the forward (FW.gRNA) or reverse (RV.gRNA) strand to the Cas9 transgene. gRNAs targeting either the EGFP coding sequence or a scrambled control were cloned into the plasmids and transfected into HEK293-EGFP cells. 48 hours post-transfection the cells were imaged using fluorescence microscopy then harvested. Relative Cas9 and gRNA expression levels were assessed with qPCR, while on-target DNA indel rates were assessed using TIDE analysis and EGFP expression was assessed with fluorescence spectroscopy and western blot.
Guide RNAs placed on the reverse (trans) strand of the plasmid resulted in a 2.3 fold increase in SaCas9 expression (n = 7, p = 0.014) and a 2.1 fold increase in gRNA expression (n = 7, p = 0.049). Increased gRNA and SaCas9 expression did not affect rates of on-target editing of EGFP. No significant differences were observed in the knockdown of levels of EGFP fluorescence or EGFP expression.
SaCas9 and gRNA expression is increased in vitro by placement of the gRNA on the reverse strand to the Cas9 unit. This may be explained by allowing better access to the U6 promoter complex required for gRNA transcription by placing it on the trans strand away from the Cas9 RNA polymerase 2 and its associated transcriptional machinery.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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