Production of gene-edited pigs harboring orthologous human mutations via
double cuttings by CRISPR/Cas9 with long single-stranded DNAs as
homology-directed repair templates by zygote injection
Precise gene edition is required for modeling human diseases in model
organisms. In this study, by using in vitro transcribed CRISPR RNA
reagents and double cuttings by CRISPR/Cas9 at two sites flanking pig
GJB2 (pGJB2) CDS with long single-stranded DNAs (lssDNA) as
homology-directed repair (HDR) templates, we generated two gene-edited
pigs of which GJB2 CDSs were replaced with a human GJB2 (hGJB2) CDS
containing c.235delC mutation and a pGJB2 CDS containing p.V37I mutation
both commonly observed in hearing loss patients, respectively.
Genotyping showed that the HDR-derived mutation efficiencies in founders
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 (ssODN) containing c.235delC mutation as HDR
template. Besides, the HDR-derived mutations were extensively integrated
into various tissues in founder and capable of germline transmission.
This study indicated that the “two cuttings with lssDNA templates”
method, which expands sgRNA selection scope and avoids direct cutting of
gene CDS, can precisely introduce human mutations into mammalian genomic
sites, especial those resistant to gene editing, with CRISPR RNAs
instead of rebonucleoproteins used in previous reports.