title: Efficient biallelic mutation in porcine parthenotes using a CRISPR-Cas9 system.
abstract: The parthenotes represent ideal models mimicking the embryonic development and characterizing the function of maternal genomes as well as an alternative source of pluripotent cell lines. Besides, parthenogenetically activated (PA) embryos serve as a rapid assay system to maximize the efficiency of generating genetically modified pig CRISPR/Cas9 system, an efficient and multiplex gene editing tool, has been utilized to modify the genome of porcine parthenotes. However, lower biallelic mutation rate and high mosaicism frequency were observed. Here, we aimed to enhance the biallelic mutation rate with reduced mosaicism by optimization of the concentration and injection time of the Cas9/sgRNA mixture in porcine parthenotes. The results showed that the efficient biallelic mutation (93%) and low mosaicism (33%) could be achieved in porcine parthenotes by cytoplasmic injection of Cas9 mRNA/sgRNA (125/12.5 ng/μl) after 8 h of parthenogenetical activation. Thus, our study provides an effective strategy for increasing the biallelic mutation rate and population homogeneity of genetically modified parthenotes, which will strengthen the role of parthenotes in uncovering early embryonic development and assessing the mutation efficiency due to the simplicity and adaptability of CRISPR/Cas9.
authors.affiliation: National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, No. 2 Yuan Ming Yuan Xi Lu, Haidian District, Beijing 100193, PR China.
authors.forename: Li
authors.initials: L
authors.lastname: Tao
authors.affiliation: National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, No. 2 Yuan Ming Yuan Xi Lu, Haidian District, Beijing 100193, PR China.
authors.forename: Mingyao
authors.initials: M
authors.lastname: Yang
authors.affiliation: National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, No. 2 Yuan Ming Yuan Xi Lu, Haidian District, Beijing 100193, PR China.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Wang
authors.affiliation: National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, No. 2 Yuan Ming Yuan Xi Lu, Haidian District, Beijing 100193, PR China.
authors.forename: Zhenni
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, No. 2 Yuan Ming Yuan Xi Lu, Haidian District, Beijing 100193, PR China.
authors.forename: Zhonghong
authors.initials: Z
authors.lastname: Wu
authors.affiliation: National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, No. 2 Yuan Ming Yuan Xi Lu, Haidian District, Beijing 100193, PR China.
authors.forename: Jianhui
authors.initials: J
authors.lastname: Tian
authors.affiliation: National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, No. 2 Yuan Ming Yuan Xi Lu, Haidian District, Beijing 100193, PR China. Electronic address: anleim@aliyun.com.
authors.forename: Lei
authors.initials: L
authors.lastname: An
authors.affiliation: National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, No. 2 Yuan Ming Yuan Xi Lu, Haidian District, Beijing 100193, PR China. Electronic address: wangshumin@cau.edu.cn.
authors.forename: Shumin
authors.initials: S
authors.lastname: Wang
mesh_terms: D000483:Alleles; D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D005260:Female; D018390:Gene Targeting; D009030:Mosaicism; D009154:Mutation; D010312:Parthenogenesis; D017393:RNA Editing; D012333:RNA, Messenger / Q000235:genetics; D013552:Swine / Q000235:genetics*
chemical_list: D012333:RNA, Messenger
keywords: Biallelic mutation; CRISPR/Cas9; Mosaicism; Parthenotes