Production of gene-edited pigs harboring orthologous human mutations via double cutting by CRISPR/Cas9 with long single-stranded DNAs as homology-directed repair templates by zygote injection.

Precise gene editing of model organisms is required for accurately modeling human diseases and deciphering gene functions. In this study, we used a pair of guide RNAs (sgRNAs), which in vitro transcribed along with other CRISPR RNA components, to generate two cleavage sites flanking pig GJB2 (pGJB2) CDS. By using long single-stranded DNAs (lssDNA) as homology-directed repair (HDR) templates, we efficiently obtained two gene-edited pigs, of which GJB2 CDS replaced with CDSs containing human GJB2 c.235delC mutation and orthologous human p.V37I mutation, respectively. These mutations were commonly observed in patients with hearing loss. Genetic analysis of the two gene-edited pigs showed that the HDR-derived gene-editing efficiency were as high as 80% (4/5) and 50% (2/4), respectively. While no mutation was observed in the group of single cutting with one sgRNA covering the 235th nucleotide C in pGJB2 CDS, using a short single-stranded oligo DNA containing c.235delC mutation as HDR template. Extra experiments proved that the intended mutations were successfully transmitted to offspring or extensively integrated into various tissues including gonad of founder pigs. Our work indicated that the new "double cutting with lssDNA template" gene editing method can expand sgRNA selection scope and avoids direct cutting of gene CDS. Additionally, can introduce precise mutations into mammalian genomic sites, especially those with unavailable proper protospacer sequence or being resistant to gene editing. Moreover, this method can be performed with CRISPR RNA reagents instead of CRISPR ribonucleoproteins applied in previous reports.