Abstract
BackgroundThe simplicity of the CRISPR/Cas9 system has enabled its widespread applications in generating animal models, functional genomic screening and in treating genetic and infectious diseases. However, unintended mutations produced by off-target CRISPR/Cas9 nuclease activity may lead to negative consequences. Especially, a very recent study found that gene editing can introduce hundreds of unintended mutations into the genome, and have attracted wide attention.ResultsTo address the off-target concerns, urgent characterization of the CRISPR/Cas9-mediated off-target mutagenesis is highly anticipated. Here we took advantage of our previously generated gene-edited sheep and performed family trio-based whole genome sequencing which is capable of discriminating variants in the edited progenies that are inherited, naturally generated, or induced by genetic modification. Three family trios were re-sequenced at a high average depth of genomic coverage (~ 25.8×). After developing a pipeline to comprehensively analyze the sequence data for de novo single nucleotide variants, indels and structural variations from the genome; we only found a single unintended event in the form of a 2.4 kb inversion induced by site-specific double-strand breaks between two sgRNA targeting sites at the MSTN locus with a low incidence.ConclusionsWe provide the first report on the fidelity of CRISPR-based modification for sheep genomes targeted simultaneously for gene breaks at three coding sequence locations. The trio-based sequencing approach revealed almost negligible off-target modifications, providing timely evidences of the safe application of genome editing in vivo with CRISPR/Cas9.
Highlights
The simplicity of the CRISPR/Cas9 system has enabled its widespread applications in generating animal models, functional genomic screening and in treating genetic and infectious diseases
We found that the rate of de novo mutations in the edited animals was equivalent to the mutation rate in human studies, suggesting the de novo single-nucleotide variants (SNV) were rather resulted from normal spontaneous mutagenesis, and not induced by genetic modification
whole genome sequencing (WGS) of three trios animal genomic DNAs yielded a total of 550 Gb of raw data, and produced between 460 and 566 million sequence reads per animal (Additional file 1: Table S1)
Summary
The simplicity of the CRISPR/Cas system has enabled its widespread applications in generating animal models, functional genomic screening and in treating genetic and infectious diseases. Wang et al BMC Genomics (2018) 19:397 since only one control animal, one guide RNA and a single CRISPR-vector for DNA injection were used [5, 6], the experiment design has very limited power to show that the magnitude of off-target effects is unusually high. This conclusion has been recently corrected by themselves when using additional mouse lines for further whole genome sequencing (WGS) analyses [7]
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