Generation of exogenous kidneys via CRISPR/Cas9 mediated blastocyst complementation targeting Osr1 gene in mice.

Blastocyst complementation has been reported to produce exogenous mouse organs including the pancreas, lungs, and kidneys, but the complemented kidneys still failed to rescue the host animals. In the present study, we generated mouse complemented kidneys through a two-step procedure: using CRISPR/Cas9 ribonucleoproteins (RNP) to knockout Osr1 alleles, followed by injecting mouse embryonic stem (ES) cells that express enhanced green fluorescent protein (EGFP). When two different sgRNAs targeting the exon 2 of Osr1 were microinjected into the pronucleus of a mouse zygote, 34% of the embryos had deletions on both alleles, and these Osr1-knockouts died with no mesonephric duct development shown by histochemical staining. With three sgRNA injections, the knockout efficiencies increased, and mesonephric duct development with EGFP-positive cells was observed in ES cell-injected E12.5 embryos. Most of the ES cell-injected Osr1-knockout embryos degenerated from E13.5 to E15.5. Four of the 264 ES cell-injected embryos were born alive and survived to the second day, with strong EGFP signals observed in both the kidneys and the heart. Therefore, complementation of the Osr1-knockout blastocyst is a potential method to produce exogenous kidneys, although further modification is still needed to increase the efficiency.