July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Validation of Staphylococcus aureus Cas9 gRNAs targeting EGFP present in the rod cells of the Nrl-EGFP mouse line.
Author Affiliations & Notes
  • Caroline Peddle
    University of Oxford, Oxford, ENGLAND, United Kingdom
  • Robert E MacLaren
    University of Oxford, Oxford, ENGLAND, United Kingdom
    Oxford Eye Hospital, Oxford, United Kingdom
  • Michelle McClements
    University of Oxford, Oxford, ENGLAND, United Kingdom
  • Footnotes
    Commercial Relationships   Caroline Peddle, None; Robert MacLaren, None; Michelle McClements, None
  • Footnotes
    Support  Mabel Churn Scholarship
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 382. doi:https://doi.org/
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      Caroline Peddle, Robert E MacLaren, Michelle McClements; Validation of Staphylococcus aureus Cas9 gRNAs targeting EGFP present in the rod cells of the Nrl-EGFP mouse line.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):382. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose : Mutations in rod cell-specific genes, such as rhodopsin, are a common cause of autosomal dominant retinitis pigmentosa. CRISPR/Cas9 gene therapy has demonstrated promise in disrupting or correcting these pathogenic mutations in vivo, often using a dual vector approach delivering the SpCas9 and gRNA on separate AAVs. Staphylococcus aureus Cas9 (SaCas9) is a species of Cas9 with a shorter, 3159 bp coding sequence, allowing delivery of both Cas9 and the gRNA on a single AAV. In this experiment SaCas9 gRNAs against the EGFP gene, present in the rod cells of the Nrl-EGFP mouse line, are validated in vitro.

Methods : The EGFP gene in the Nrl-EGFP mice was sequenced and screened for SaCas9 gRNA sequences. Six gRNAs were selected for in vitro validation and cloned into a plasmid expressing both SaCas9 and the gRNA sequence. Two control plasmids were created, a “scrambled” control containing a nonsense gRNA, and a “no-PAM” control, containing a gRNA homologous to EGFP but with no PAM site present in the DNA. These plasmids were transfected into HEK293-EGFP cells. 48 hours post-transfection the cells were imaged using fluorescence microscopy then harvested. The reduction in EGFP expression was quantified using mean grey value, fluorescence spectroscopy and qPCR. The presence of DNA insertions and deletions at each gRNA target site was confirmed using the T7E1 assay.

Results : All six gRNAs screened resulted in detectable DNA disruption in HEK293-EGFP cells using the T7E1 assay, with no disruption seen using either control gRNA (scrambled and no-PAM). The highest performing gRNA, F13, resulted in over 50% reduction in EGFP mRNA levels compared to the negative control (One-way ANOVA P ≤ 0.005). Both the scrambled and no-PAM control had no significant reduction in mean grey value, EGFP fluorescence levels, or EGFP mRNA levels.

Conclusions : SaCas9 was successfully used to knock-down EGFP in vitro using a single construct expressing both SaCas9 and the gRNA. Several potential gRNAs were identified which could be used in vivo in a single AAV construct against the Nrl-EGFP mouse line to demonstrate the use of this technique for targeting pathogenic rod cell mutations.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.


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