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Screening of CRISPR/Cas base editors to target the AMD high-risk Y402H complement factor H variant

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Nguyen Tran, MT, Mohd Khalid, MKN, Pebay, A, Cook, AL ORCID: 0000-0003-1770-7910, Liang, HH, Wong, RCB, Craig, JE, Liu, G-S ORCID: 0000-0003-3379-724X, Hung, SS and Hewitt, AW ORCID: 0000-0002-5123-5999 2019 , 'Screening of CRISPR/Cas base editors to target the AMD high-risk Y402H complement factor H variant' , Molecular Vision, vol. 25 , pp. 174-182 .

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Abstract

Purpose: 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.Methods: 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.Results: 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).Conclusions: 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.

Item Type: Article
Authors/Creators:Nguyen Tran, MT and Mohd Khalid, MKN and Pebay, A and Cook, AL and Liang, HH and Wong, RCB and Craig, JE and Liu, G-S and Hung, SS and Hewitt, AW
Journal or Publication Title: Molecular Vision
Publisher: Molecular Vision
ISSN: 1090-0535
Copyright Information:

Copyright 2019 Molecular Vision. Licensed under Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported (CC BY-NC-ND 3.0) https://creativecommons.org/licenses/by-nc-nd/3.0/

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