June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Genomic ablation of rhesus macaque VEGFa using CRISPR-Cas9 ribonucleoprotein
Author Affiliations & Notes
  • Tzu-Ni Sin
    University of California Davis, Davis, California, United States
  • Sook Hyun Chung
    University of California Davis, Davis, California, United States
  • Taylor Ngo
    University of California Davis, Davis, California, United States
  • Glenn Yiu
    University of California Davis, Davis, California, United States
  • Footnotes
    Commercial Relationships   Tzu-Ni Sin, None; Sook Hyun Chung, None; Taylor Ngo, None; Glenn Yiu, Allergan (C), Carl Zeiss Meditec (C), Clearside Biomedical (C), Genentech (C), Intergalactic Therapeutics (C), Iridex (C), Regeneron (C), Topcon (C), Verily (C)
  • Footnotes
    Support  NIH K08 EY026101 and NIH R21 EY031108
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 1463. doi:
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      Tzu-Ni Sin, Sook Hyun Chung, Taylor Ngo, Glenn Yiu; Genomic ablation of rhesus macaque VEGFa using CRISPR-Cas9 ribonucleoprotein. Invest. Ophthalmol. Vis. Sci. 2021;62(8):1463.

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

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Purpose : Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing technology has significant potential as a novel mode of gene therapy for retinal disorders. We previously identified single guide RNAs (gRNAs) targeting conserved regions in exon 1 of the vascular endothelial growth factor-a (VEGFA) gene in mice, rhesus macaques, and humans, and demonstrated efficient genomic ablation in human cell lines in vitro and mouse eyes in vivo using S. pyogenes Cas9 (SpCas9). Here, we evaluate the effectiveness of these gRNAs to target VEGFA in the rhesus macaque genome using SpCas9 ribonucleoproteins (RNPs) in a cell-free system.

Methods : We identified 2 target sequences (V1 and V2) in exon 1 of mouse, rhesus macaque, and human VEGFA gene in silico, then synthetized functional gRNAs by combining these target sequences with trans-activating crispr RNA (tracrRNA) and assembled with SpCas9 protein to generate VEGFA-targeting RNPs. The 1.4kb target VEGFA gene was amplified from rhesus macaque and human genomic DNA separately, then incubated with V1, V2, or both V1+V2 RNPs to compare genome editing efficiency in vitro.

Results : The in silico predictions for on-target activity of V1 and V2 target sequences were 60.5 and 49.5 for rhesus VEGFa, and 60.0 and 54.0 for human VEGFa, respectively. The off-target scores were 43.5 and 45.5 for rhesus VEGFa and 43.0 and 45.0 for human VEGFa. RNPs targeting the V1 and V2 loci of VEGFA demonstrated successful genomic ablation of the target gene sequence of rhesus macaques in vitro, with a genome editing efficiency of 46.39% using V1 gRNAs, 20.95% using V2 gRNAs, and 44.60% using V1+V2 gRNAs. In comparison, genome editing efficiencies using human VEGFA, which has similar on- and off-target scores to those of rhesus macaque, were 84.72% using V1 gRNAs, 37.10% using V2 gRNAs, and 68.54% using V1+V2 gRNAs.

Conclusions : CRISPR-based genomic ablation of the VEGFA gene using SpCas9 RNPs can be achieved with nonhuman primate and human genomes in vitro, although genome editing efficiencies do not reflect in silico predictions. In vitro assays may better indicate in vivo effectiveness of VEGFA ablation than in silico predictions, and may help facilitate clinical translation of CRISPR-based anti-angiogenesis therapies.

This is a 2021 ARVO Annual Meeting abstract.


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