Fig. 4

CRISPR-Cas9 editing in porcine PSCs and PSCdMs. A IRF3 CRISPR/Cas9 editing diagram. The entire IRF3 coding sequence was deleted using a pair of guides (blue lightning bolts) designed to cut immediately after the initiation codon and 3 bp upstream of the stop codon. Coding exons are shown as red boxes, non-coding genomic sequence as thick black lines and 5′ and 3′ UTRs as brown boxes. Genotyping was performed using a pool of two forward primers (green and yellow arrowheads) and one reverse primer (pink arrowhead), where the yellow forward primer located in exon 6 is specific for the wild-type allele. PCR product sizes are indicated. B PCR analysis showing the expected products for three wild-type (WT) and three IRF3 knock-out (KO) clones using the primer pool indicated in A. Water (-ve) and parental porcine PSC genomic DNA (+ve) were included as controls. C Bright-field images of PSCdM generated from wild-type (WT) and IRF3 knock-out (KO) clones. D RT-qPCR analysis for IRF3 expression in PSCdMs from wild-type (WT) and IRF3 knock-out (KO) clones. Mean and SD of three biological replicates. E Ratio of PRRSV-infected WT and IRF3 KO porcine PSCdMs in poly(I:C)-treated:untreated conditions. Data represents ratio of parental line and mean ratio of three KO clones. F Brightfield and fluorescent images of lenti-EGFP-transduced porcine PSCdMs, 7 d post-transduction. G Flow cytometry analysis of lenti-EGFP-transduced porcine PSCdMs 7 d post-transduction relative to non-transduced porcine PSCdMs. H CD163 CRISPR/Cas9 editing diagram. A pair of guides that delete exon7 [41] results in a 487 bp deletion that can be detected by PCR using flanking primers (green and pink arrowheads). Expected PCR product sizes are indicated. I PCR analysis demonstrating lentiviral-mediated CD163 editing in porcine PSCdMs using the screening strategy in panel H. Water (-ve) and non-transduced cells (Non) were included as controls