Efficient biallelic mutation in porcine parthenotes using a CRISPR-Cas9 system.
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.
['Tao|Li|L|', 'Yang|Mingyao|M|', 'Wang|Xiaodong|X|', 'Zhang|Zhenni|Z|', 'Wu|Zhonghong|Z|', 'Tian|Jianhui|J|', 'An|Lei|L|', 'Wang|Shumin|S|']
D000483:Alleles D000818:Animals D030801:Animals, Genetically Modified 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 D013552:Swine
Biallelic mutation Parthenotes CRISPR/Cas9 Mosaicism