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

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

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)174-182
Number of pages9
JournalMolecular Vision
Volume25
Publication statusPublished - 16 Mar 2019

Keywords

  • ge-related macular degeneration
  • CRISPR
  • Complement factor H
  • opthamology

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    Tran, M. T. N., Khalid, M. K. N. M., Pébay, A., Cook, A. L., Liang, H. H., Wong, R. C. B., Craig, J. E., Liu, G-S., Hung, S. S., & Hewitt, A. W. (2019). Screening of CRISPR/Cas base editors to target the AMD high-risk Y402H complement factor H variant. Molecular Vision, 25, 174-182. http://www.molvis.org/molvis/v25/174