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Alex W Hewitt, Peter Tran, Mohd Khairul Nizam Mohd Khalid, Alice Pébay, Anthony L Cook, Helena Liang, Raymond Wong, Jamie E Craig, Guei-Sheung Liu, Sandy Hung; CRISPR/Cas-mediated base editing of the AMD high-risk Y402H complement factor H variant. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4947. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the efficacy of using a CRISPR/Cas-mediated strategy to correct a common high-risk allele that is associated with age-related macular degeneration (AMD) (rs1061170; NM_000186.3:c.1204T>C; NP_000177.2:p.His402Tyr) in the complement factor H (CFH) gene.
A human embryonic kidney cell line (HEK293A) was engineered to contain the pathogenic risk variant for AMD (HEK293A-CFH). Several different base editor constructs (BE3, SaBE3, SaKKH-BE3, VQR-BE3, and Target-AID) and their respective single-guide RNA (sgRNA)-expression cassettes targeting either the pathogenic risk variant allele in the CFH locus or the LacZ gene, as a negative control, were evaluated head-to-head for the incidence of a cytosine-to-thymine nucleotide correction. The base editor construct that showed appreciable editing activity was selected for further assessment in which the base-edited region was subjected to next-generation deep sequencing to quantify on-target and off-target editing efficacy.
The tandem use of the Target-AID base editor and its respective sgRNA demonstrated a base editing efficiency of facilitating a cytosine-to-thymine nucleotide correction in 21.5% of the total sequencing reads. Additionally, the incidence of insertions and deletions (indels) was detected in only 0.15% of the sequencing reads, with virtually no off-target effects evident across the top 11 predicted off-target sites containing at least one cytosine in the activity window (n=3, pooled amplicons).
CRISPR-mediated base editing can be used to facilitate a permanent and stably inherited cytosine-to-thymine nucleotide correction of the rs1061170 SNP in the CFH gene with minimal off-target effects.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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