Validation of reliable safe harbor locus for efficient porcine transgenesis

Transgenic technology is now widely used in biomedical and agricultural fields. Transgenesis is commonly achieved through random integration which might cause some uncertain consequences. The site-specific integration could avoid this disadvantage. This study aimed to screen and validate the best safe harbor (SH) locus for efficient porcine transgenesis. First, the cells carrying the EGFP reporter construct at four different SH loci (ROSA26, AAVS1, H11 and COL1A1) were achieved through CRSIPR/Cas9-mediated HDR. At the COL1A1 and ROSA26 loci, a higher mRNA and protein expression of EGFP was detected, and it was correlated with a lower level of DNA methylation of the EGFP promoter, hEF1α. A decreased H3K27me3 modification of the hEF1α promoter at the COL1A1 locus was also detected. For the safety of transgenesis at different SH locus, we found that transgenesis could relatively alter the expression of the adjacent endogenous genes, but the influence was limited. We also did not observe any off-target cleavage for the selected sgRNAs of the COL1A1 and ROSA26 loci. In conclusion, the COL1A1 and ROSA26 were confirmed to be the best two SH loci with the COL1A1 being more competitive for porcine transgenesis. This work would greatly facilitate porcine genome engineering and transgenic pig production.