title: Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs.
abstract: Genetic engineering in livestock was greatly enhanced by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), which can be programmed with a single-guide RNA (sgRNA) to generate site-specific DNA breaks. However, the uncertainties caused by wide variations in sgRNA activity impede the utility of this system in generating genetically modified pigs. Here, we described a single blastocyst genotyping system to provide a simple and rapid solution to evaluate and compare the sgRNA efficiency at inducing indel mutations for a given gene locus. Assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days from the design of the sgRNA. The most effective sgRNA selected by this system was successfully used to induce site-specific insertion through homology-directed repair at a frequency exceeding 13%. Additionally, the highly efficient gene deletion via the selected sgRNA was confirmed in pig fibroblast cells, which could serve as donor cells for somatic cell nuclear transfer. We further showed that direct cytoplasmic injection of Cas9 mRNA and the favorable sgRNA into zygotes could generate biallelic knockout piglets with an efficiency of up to 100%. Thus, our method considerably reduces the uncertainties and expands the practical possibilities of CRISPR/Cas9-mediated genome engineering in pigs.
authors: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
authors: Xianlong
authors: X
authors: Wang
authors: College of Veterinary Medicine, Sichuan Agriculture University, Ya'an, Sichuan 625014, China.
authors: Jinwei
authors: J
authors: Zhou
authors: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
authors: Chunwei
authors: C
authors: Cao
authors: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
authors: Jiaojiao
authors: J
authors: Huang
authors: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
authors: Tang
authors: T
authors: Hai
authors: Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
authors: Yanfang
authors: Y
authors: Wang
authors: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
authors: Qiantao
authors: Q
authors: Zheng
authors: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
authors: Hongyong
authors: H
authors: Zhang
authors: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
authors: Guosong
authors: G
authors: Qin
authors: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
authors: Xiangnan
authors: X
authors: Miao
authors: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
authors: Hongmei
authors: H
authors: Wang
authors: College of Veterinary Medicine, Sichuan Agriculture University, Ya'an, Sichuan 625014, China.
authors: Suizhong
authors: S
authors: Cao
authors: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
authors: Qi
authors: Q
authors: Zhou
authors: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
authors: Jianguo
authors: J
authors: Zhao
mesh_terms: D000483:Alleles; D000818:Animals; D001483:Base Sequence; D001755:Blastocyst / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000235:genetics*; D005347:Fibroblasts / Q000378:metabolism; D055879:Gene Knock-In Techniques; D055786:Gene Knockout Techniques; D018390:Gene Targeting; D060005:Genotyping Techniques; D051739:Microphthalmia-Associated Transcription Factor / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation / Q000235:genetics; D012150:Polymorphism, Restriction Fragment Length; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034421:Sus scrofa / Q000235:genetics*; D015053:Zygote / Q000378:metabolism
chemical_list: D051739:Microphthalmia-Associated Transcription Factor; D000094704:RNA, Guide, CRISPR-Cas Systems; D012333:RNA, Messenger