Abstract
The introduction of frameshift indels by genome editing has emerged as a powerful technique to study the functions of uncharacterized genes in cell lines and model organisms. Such mutations should lead to mRNA degradation owing to nonsense-mediated mRNA decay or the production of severely truncated proteins. Here, we show that frameshift indels engineered by genome editing can also lead to skipping of “multiple of three nucleotides” exons. Such splicing events result in in-frame mRNA that may encode fully or partially functional proteins. We also characterize a segregating nonsense variant (rs2273865) located in a “multiple of three nucleotides” exon of LGALS8 that increases exon skipping in human erythroblast samples. Our results highlight the potentially frequent contribution of exonic splicing regulatory elements and are important for the interpretation of negative results in genome editing experiments. Moreover, they may contribute to a better annotation of loss-of-function mutations in the human genome.
Highlights
The success of genome-wide association studies (GWAS) in discovering DNA sequence variants associated with common human diseases and other complex traits has highlighted the challenge to characterize the functions of novel genes
In an attempt to study the role of phosphatase and actin regulator 1 (PHACTR1), a gene implicated in coronary artery disease by GWAS [17, 18], we used clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 to introduce frameshift indels in its first three translated exons in the teloHAEC cell line, and isolated four clones (S1 Table)
We anticipated that frameshift indels in exons 9 and 10, which are sufficiently distant from the start codon and the last exon, would reduce PHACTR1 expression owing to nonsense-mediated mRNA decay (NMD) [19]
Summary
The success of genome-wide association studies (GWAS) in discovering DNA sequence variants associated with common human diseases and other complex traits has highlighted the challenge to characterize the functions of novel genes. Traditional strategies require gene knockdown using short-interfering RNA (siRNA) or over-expression with ectopic vectors in relevant cell types. Powerful, these methods have their own caveats: siRNA can have off-target effects and over-expression can lead to non-physiological levels of gene expression. Zinc finger nucleases and transcription activator-like effector nucleases (TALEN) were the initial tools of genome editing, but their use was limited by the complexity to target their nuclease activity to the loci of interest.
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