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

Citation

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, Screening of CRISPR/Cas base editors to target the AMD high-risk Y402H complement factor H variant, Molecular Vision, 25 pp. 174-182. ISSN 1090-0535 (2019) [Refereed Article]


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Copyright Statement

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/

Official URL: http://www.molvis.org/molvis/v25/174/

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 Details

Item Type:Refereed Article
Research Division:Medical and Health Sciences
Research Group:Ophthalmology and Optometry
Research Field:Vision Science
Objective Division:Health
Objective Group:Clinical Health (Organs, Diseases and Abnormal Conditions)
Objective Field:Hearing, Vision, Speech and Their Disorders
UTAS Author:Nguyen Tran, MT (Mr Minh Nguyen Tran)
UTAS Author:Mohd Khalid, MKN (Mr Mohd Mohd Khalid)
UTAS Author:Cook, AL (Associate Professor Tony Cook)
UTAS Author:Liu, G-S (Dr Guei-Sheung Liu)
UTAS Author:Hewitt, AW (Professor Alex Hewitt)
ID Code:132604
Year Published:2019
Funding Support:National Health and Medical Research Council (APP1103329)
Deposited By:Wicking Dementia Research and Education Centre
Deposited On:2019-05-15
Last Modified:2019-08-21
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