Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off-target effects

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Abstract

Background: The CRISPR-Cas9 system is a widely utilized platform for transgenic animal production in various species, although its off-target effects should be addressed. Several applications of this tool have been proposed in model animals but remain insufficient for transgenic livestock production. Results: Here, we report the first application of single Cas9 nickase (Cas9n) to induce gene insertion at a selected locus in cattle. We identify the main binding sites of a catalytically inactive Cas9 (dCas9) protein in bovine fetal fibroblast cells (BFFs) with chromatin immunoprecipitation sequencing (ChIP-seq). Subsequently, we demonstrate that a single Cas9n-induced single-strand break can stimulate the insertion of the natural resistance-associated macrophage protein-1 (NRAMP1) gene with reduced, but still considerable, off-target effects. Through somatic cell nuclear transfer, we finally obtain transgenic cattle with increased resistance to tuberculosis. Conclusions: Our results contribute to the development of CRISPR-Cas9 system for agriculture applications.

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Gao, Y., Wu, H., Wang, Y., Liu, X., Chen, L., Li, Q., … Zhang, Y. (2017). Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off-target effects. Genome Biology, 18(1). https://doi.org/10.1186/s13059-016-1144-4

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