Generation of GGTA1 Mutant Pigs by Direct Pronuclear Microinjection of CRISPR/Cas9 Plasmid Vectors

Abstract

This study was conducted to confirm that 1-site and 4-site ppU6-GGTA1-gRNA CRISPR vectors together with the pCX-Flag2-NLS-Cas9-NLS plasmid 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-NLS-Cas9-NLS plasmid 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.

Lessening of porcine epidemic diarrhoea virus susceptibility in piglets after editing of the CMP-N-glycolylneuraminic acid hydroxylase gene with CRISPR/Cas9 to nullify N-glycolylneuraminic acid expression

“…We have established TALEN [26] and CRISPR/Cas9 [27, 28] gene editing techniques in which the editing plasmid vector is directly microinjected into the pronucleus of newly fertilized eggs and have used these techniques to generate GGTA1 mutant pigs for the study of xenotransplantation. In this study, two sgRNAs directed against exon II and intron 2 of the CMAH gene, together with Cas9 mRNA, were microinjected into the cytoplasm near the pronucleus; the microinjected eggs yielded 4 live pigs and one stillborn pig carrying a null function of the CMAH gene.…”

“…Furthermore, GE can be simultaneously conducted on multiple sites or genes with the same Cas9 to achieve different targeting purposes or reduce the risks of off-targeting [23–25]. We have established TALEN [26] and CRISPR/Cas9 [27, 28] systems for direct microinjection of GE vectors to generate GGTA1 mutant pigs. In this study, direct microinjection of two single-guide RNA and Cas9 mRNA vectors into the cytoplasm of pronuclear porcine embryos was used to generate CMAH mutant pigs with null expression of NGNA, and the possibility of obtaining mutant piglets that are resistant to infection by PEDV was examined.…”

The Scaffold Derived from GGTA1 and CMAH Gene Knockout Pigs Generated by Gene Editing Provoked Little Inflammatory Responses in a Humanized Pig Model

“…The GGTA1 KO and CMAH KO pigs were generated by Chung et al [12] and Tu et al [13], respectively, by CRISPR/Cas9 gene editing. In this study, the animals were generated by cross breeding GGTA1 KO boars with CMAH KO sows.…”

“…Depleting α-Gal and converting Neu5Gc to Neu5Ac of human form to diminish the antigenicity can be achieved only by knocking out the responsible genes in the pig genome. The α-Gal free or GGTA1 knockout (KO) pigs [12] and Neu5Ac (Neu5Gc free) or CMAH KO pigs [13] have already been generated through our continuous efforts with adopted CRISP/Cas9 gene editing techniques. Thus, the humanized porcine (HP) SIS-ECM that can be obtained from the dKO pigs can also serve as an animal model for antigenicity testing.…”

CRISPR is knocking on barn door

“…CRISPR can be also delivered as ribonucleoprotein (Park, Powell, et al., ; Sheets et al., ), with the advantage that it acts faster than RNA delivery, as it does not require the generation of Cas9 protein by the zygote. The components can be also delivered as plasmid (Chuang et al., ; Honda et al., ; Petersen et al., ), but this is the slowest way, as Cas9 needs to be transcribed and translated, and it entails a prolonged presence of CRISPR components that favours the appearance of off‐target effects (OTEs). OTEs were initially an important matter of concern for CRISPR editing, but whole‐genome sequencing analysis of cells constitutively expressing CRISPR components (i.e.…”