Enhanced knockout and knock-in efficiency through coselection strategy. (A) Generation of PFF clones with GGTA1 and CMAH knockouts, and hTHBD and hCD55 knock-ins via Cas9-mediated NHEJ. The target sites, located in the first exon of GGTA1 and CMAH, are indicated by arrows. Puro and Neo serve as surrogate reporters for GGTA1 and CMAH targeting, respectively. The EF1a-hTHBD-P2A-hCD55-pA sequence is used for insertion. Clones are categorized as follows: (Homo + Hete) one gene homozygously edited, one heterozygously edited in GGTA1 and CMAH, (Homo) both genes homozygously edited, and (Homo + KI) both genes homozygously edited with hTHBD and hCD55 knock-ins. (B) Generation of PFF clones with triple knockout of GGTA1, B4GALNT2, and CMAH and with knock-in of six genes—hHOMX1, hCD55, hCD47, hCD46, hTHBD, and hEPCR—via Cas9-mediated HDR. V2.1G is used for B4GALNT2 targeting. The CMV-hHOMX1-T2A-hCD55-EF1a-hCD47-P2A-hCD46-pA cassette is inserted into the GGTA1 locus, and the ICAM2-hTHBD-P2A-hEPCR-pA cassette is inserted into the CMAH locus, using ∼1 kb of homologous arms. (Right) Junction PCR for V2.1G-selected (V; V2.1G) and control (C; control) cell pools. Control pools consist of transfected positive cells. Primers detect left and right junctions in CMAH and GGTA1 loci, with “GAPDH” as a normalization control. (C) Analysis of triple gene knockout in “3KO + 6KI” cell pools. (D) Ratio of triple knockout and six genes knock-in PFF clones in V2.1G-selected and control groups. BM-PFFs♂ and BM-PFFs♀ represent PFFs from male and female Bama miniature pigs, respectively. WZS-PFFs♂ indicates PFFs from male Wuzhishan pigs.
