Low incidence of SNVs and indels in trio genomes of Cas9-mediated multiplex edited sheep
Abstract
Background: 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. 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. Results: To 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. Conclusions: We 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.
Publication Title
BMC Genomics
Recommended Citation
Wang, X., Liu, J., Niu, Y., Li, Y., Zhou, S., Li, C., Ma, B., Kou, Q., Petersen, B., Sonstegard, T., Huang, X., Jiang, Y., & Chen, Y. (2018). Low incidence of SNVs and indels in trio genomes of Cas9-mediated multiplex edited sheep. BMC Genomics, 19 (1) https://doi.org/10.1186/s12864-018-4712-z
