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

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.

• Authors: Minh Thuan Nguyen Tran, Mohd Khairul Nizam Mohd Khalid, Alice Pébay, Anthony L Cook, Helena H Liang, Raymond C B Wong, Jamie E Craig, Guei-Sheung Liu, Sandy S Hung, Alex W Hewitt
• Journal: Molecular vision (Mol Vis) Vol. 25 Pg. 174-182 ( 2019) ISSN: 1090-0535 [Electronic] United States
• PMID: 30996586 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• CFH protein, human
• RNA, Guide, CRISPR-Cas Systems
• Complement Factor H
• Cytosine
• CRISPR-Associated Protein 9
• Thymine

Topics

• Base Sequence
• CRISPR-Associated Protein 9 (genetics, metabolism)
• CRISPR-Cas Systems
• Clustered Regularly Interspaced Short Palindromic Repeats
• Complement Factor H (genetics, metabolism)
• Cytosine (metabolism)
• Gene Editing (methods)
• Gene Expression
• HEK293 Cells
• High-Throughput Nucleotide Sequencing
• Humans
• Lac Operon
• Macular Degeneration (genetics, metabolism, pathology)
• Mutation
• Plasmids (chemistry, metabolism)
• RNA, Guide, CRISPR-Cas Systems (genetics, metabolism)
• Thymine (metabolism)