Screening of CRISPR/Cas base editors to target the AMD high-risk Y402H complement factor H variant
AuthorMinh, TNT; Khalid, MKNM; Pebay, A; Cook, AL; Liang, HH; Wong, RCB; Craig, JE; Liu, G-S; Hung, SS; Hewitt, AW
Source TitleMOLECULAR VISION
University of Melbourne Author/sPebay, Alice; Liang, Helena; Wong, Raymond; HEWITT, ALEXANDER; Liu, Guei-Sheung; Craig, Jamie; Hung, Sandy
Document TypeJournal Article
CitationsMinh, T. N. T., Khalid, M. K. N. M., Pebay, 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, pp.174-182
Access StatusOpen Access
Open Access at PMChttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441356
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.
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