BACKGROUND: The recent development of the type II clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has enabled genome editing of mammalian genomes including those of mice and human; however, its applicability and efficiency in the pig have not been studied in depth. Here, using the CRISPR/Cas9 system, we aimed to destroy the function of the porcine α-1,3-galactosyltransferase (α-GalT) gene (GGTA1) whose product is responsible for the synthesis of the α-Gal epitope, a causative agent for hyperacute rejection upon pig-to-human xenotransplantation. METHODS: Porcine embryonic fibroblasts were transfected with a Cas9 expression vector and guide RNA specifically designed to target GGTA1. At 4 days after transfection, the cells were incubated with IB4 conjugated with saporin (IB4SAP), which eliminates α-Gal epitope-expressing cells. Therefore, the cells surviving after IB4SAP treatment would be those negative for α-Gal epitope expression, which in turn indicates the generation of GGTA1 biallelic knockout (KO) cells. RESULTS: Of the 1.0 × 10(6) cells transfected, 10-33 colonies survived after IB4SAP treatment, and almost all colonies (approximately 90%) were negative for staining with red fluorescence-labeled IB4. Sequencing of the mutated portion of GGTA1 revealed a frameshift of the α-GalT protein. Porcine blastocysts derived from the somatic cell nuclear transfer of these α-Gal epitope-negative cells also lacked the α-Gal epitope on their surface. CONCLUSIONS: These results demonstrated that the CRISPR/Cas9 system can efficiently induce the biallelic conversion of GGTA1 in the resulting somatic cells and is thus a promising tool for the creation of KO cloned piglets.
BACKGROUND: The recent development of the type II clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has enabled genome editing of mammalian genomes including those of mice and human; however, its applicability and efficiency in the pig have not been studied in depth. Here, using the CRISPR/Cas9 system, we aimed to destroy the function of the porcine α-1,3-galactosyltransferase (α-GalT) gene (GGTA1) whose product is responsible for the synthesis of the α-Gal epitope, a causative agent for hyperacute rejection upon pig-to-human xenotransplantation. METHODS: Porcine embryonic fibroblasts were transfected with a Cas9 expression vector and guide RNA specifically designed to target GGTA1. At 4 days after transfection, the cells were incubated with IB4 conjugated with saporin (IB4SAP), which eliminates α-Gal epitope-expressing cells. Therefore, the cells surviving after IB4SAP treatment would be those negative for α-Gal epitope expression, which in turn indicates the generation of GGTA1 biallelic knockout (KO) cells. RESULTS: Of the 1.0 × 10(6) cells transfected, 10-33 colonies survived after IB4SAP treatment, and almost all colonies (approximately 90%) were negative for staining with red fluorescence-labeled IB4. Sequencing of the mutated portion of GGTA1 revealed a frameshift of the α-GalT protein. Porcine blastocysts derived from the somatic cell nuclear transfer of these α-Gal epitope-negative cells also lacked the α-Gal epitope on their surface. CONCLUSIONS: These results demonstrated that the CRISPR/Cas9 system can efficiently induce the biallelic conversion of GGTA1 in the resulting somatic cells and is thus a promising tool for the creation of KO cloned piglets.
Authors: Jose L Estrada; Greg Martens; Ping Li; Andrew Adams; Kenneth A Newell; Mandy L Ford; James R Butler; Richard Sidner; Matt Tector; Joseph Tector Journal: Xenotransplantation Date: 2015-03-01 Impact factor: 3.907
Authors: Siddharth Patel; Avathamsa Athirasala; Paula P Menezes; N Ashwanikumar; Ting Zou; Gaurav Sahay; Luiz E Bertassoni Journal: Tissue Eng Part A Date: 2018-06-07 Impact factor: 3.845