title: Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off-target effects.
abstract: 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.
  
  
  
  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.
  
  
  
  Our results contribute to the development of CRISPR-Cas9 system for agriculture applications.
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, 712100, Shaanxi, China.
authors.forename: Yuanpeng
authors.initials: Y
authors.lastname: Gao
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, 712100, Shaanxi, China.
authors.forename: Haibo
authors.initials: H
authors.lastname: Wu
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, 712100, Shaanxi, China.
authors.forename: Yongsheng
authors.initials: Y
authors.lastname: Wang
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, 712100, Shaanxi, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Liu
authors.affiliation: Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, 712100, Shaanxi, China.
authors.forename: Linlin
authors.initials: L
authors.lastname: Chen
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, 712100, Shaanxi, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Li
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, 712100, Shaanxi, China.
authors.forename: Chenchen
authors.initials: C
authors.lastname: Cui
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, 712100, Shaanxi, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Liu
authors.affiliation: Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, 712100, Shaanxi, China.
authors.forename: Jingcheng
authors.initials: J
authors.lastname: Zhang
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China. zhy1956@263.net.|Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, 712100, Shaanxi, China. zhy1956@263.net.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001665:Binding Sites; D064113:CRISPR-Cas Systems; D027682:Cation Transport Proteins / Q000235:genetics*; D002417:Cattle; D003850:Deoxyribonuclease I / Q000378:metabolism*; D060467:Disease Resistance / Q000235:genetics; D055879:Gene Knock-In Techniques; D023061:Gene Order; D018390:Gene Targeting; D056426:Genetic Loci; D005822:Genetic Vectors / Q000235:genetics; D016254:Mutagenesis, Insertional; D053652:Nuclear Transfer Techniques; D011485:Protein Binding; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D059767:Recombinational DNA Repair; D019076:Transgenes; D000099173:Natural Resistance-associated Macrophage Protein 1
chemical_list: D027682:Cation Transport Proteins; D003850:Deoxyribonuclease I; D000094704:RNA, Guide, CRISPR-Cas Systems; D000099173:Natural Resistance-associated Macrophage Protein 1
keywords: CRISPR-Cas9; Chromatin immunoprecipitation sequencing (ChIP-seq); Homologous recombination; Nickase; Off-target; Single-strand break; Tuberculosis