Generation of GGTA1 Mutant Pigs by Direct Pronuclear Microinjection of CRISPR/Cas9 Plasmid Vectors.
This study was conducted to confirm that 1-site and 4-site ppU6-GGTA1-gRNA CRISPR vectors together with the pCX-Flag2-NLS1-Cas9-NLS2plasmid can both generate KO pigs by direct pronuclear microinjection. In total, 41 and 53 fertilized eggs were microinjected on 1-site and 4-site strategies, respectively. The 1-site construction generated a litter of 8 piglets, and 2 were mono-allelic mutant (mMt). The injection of 4-site constructions resulted in one biallelic mutant (bMt) and one mMt piglet in a litter of 7. Those 3 mMt pigs had a 4 bp deletion, 5 bp insertion, or 7 bp insertion at site I, and the bMt pig had 5 types of mutations at cleavage sites I and III. The expression of alpha-Gal on the bMt peripheral blood mononuclear cells (PBMCs) was reduced, and survival rate of bMt PBMCs was maintained as indicated by results of cultivation with sera of humans or Formosan Macaques. We concluded that mutant pigs could be generated by direct pronuclear microinjection of ppU6-GGTA1-gRNA CRISPR vectors with the pCX-Flag2-NLS1-Cas9-NLS2plasmid and that the 4-site strategy has a better mutant efficiency. Porcine U6 promoter was firstly used to express KO vectors and effectively generate mutant pigs, worthily to adopt for future KO studies.
a Division of Animal Technology, Animal Technology Laboratories , Agricultural Technology Research Institute , Taiwan , Republic of China.
Chin-Kai
CK
Chuang
a Division of Animal Technology, Animal Technology Laboratories , Agricultural Technology Research Institute , Taiwan , Republic of China.
Chien-Hong
CH
Chen
a Division of Animal Technology, Animal Technology Laboratories , Agricultural Technology Research Institute , Taiwan , Republic of China.
Chung-Ling
CL
Huang
a Division of Animal Technology, Animal Technology Laboratories , Agricultural Technology Research Institute , Taiwan , Republic of China.
Yu-Hsiu
YH
Su
a Division of Animal Technology, Animal Technology Laboratories , Agricultural Technology Research Institute , Taiwan , Republic of China.
Shu-Hui
SH
Peng
a Division of Animal Technology, Animal Technology Laboratories , Agricultural Technology Research Institute , Taiwan , Republic of China.
Tai-Yun
TY
Lin
b Department of Surgery , National Taiwan University Hospital , Taipei, Taiwan , Republic of China.
Hao-Chih
HC
Tai
c Department of Biotechnology and Animal Science , National Ilan University , Taiwan , Republic of China.
Tien-Shuh
TS
Yang
a Division of Animal Technology, Animal Technology Laboratories , Agricultural Technology Research Institute , Taiwan , Republic of China.
Ching-Fu
CF
Tu
D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D005260:Female; D005700:Galactosyltransferases / Q000235:genetics*; D055786:Gene Knockout Techniques / Q000379:methods*; D018014:Gene Transfer Techniques; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008297:Male; D008845:Microinjections; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D013552:Swine
D005700:Galactosyltransferases; C530267:alpha-1,3-galactosyltransferase 1, porcine
CRISPR/Cas9 KO Plasmid; GGTA1 mutant pigs; RNA polymerase III promoter; pronuclear microinjection