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Mol Vis. 2019 Mar 16;25:174-182. eCollection 2019.

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

Author information

1
Menzies Institute for Medical Research, University of Tasmania, Tasmania, Australia.
2
Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, Victoria, Australia.
3
Ophthalmology, Department of Surgery, University of Melbourne, Victoria, Australia.
4
Department of Anatomy and Neuroscience, University of Melbourne, Victoria, Australia.
5
Wicking Dementia Research and Education Centre, University of Tasmania, Hobart, TAS 7000, Australia.
6
Department of Ophthalmology, Flinders University, Flinders Medical Centre, Bedford Park, Australia.

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.

PMID:
30996586
PMCID:
PMC6441356

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