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Updated: Aug 2, 2026
Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System
Published on: May 28, 2015
Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off-target effects
Yuanpeng Gao1,2, Haibo Wu1,2, Yongsheng Wang1,2
1College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.
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.
