Cloning and base editing of GFP transgenic rhesus monkey and off-target analysis.

We report the cloning of a 12-year-old transgenic green fluorescent protein (GFP) monkey by somatic cell nuclear transfer (SCNT) and base editing of the embryos, accompanied with safety evaluation of adenine base editors (ABEs). We first show the ability of ABEmax to silence GFP through A-to-G editing of the GFP sequence in 293T cells. Subsequently, using donor cells from a monkey expressing GFP, we have successfully generated 207 ABEmax-edited (SCNT-ABE) and 87 wild-type (SCNT) embryos for embryo transfer, genotyping, and genome and transcriptome analysis. SCNT-ABE and SCNT embryos are compared for off-target analysis without the interference of genetic variants using a new method named as OA-SCNT. ABEmax does not induce obvious off-target DNA mutations but induces widespread off-target RNA mutations, 35% of which are exonic, in edited monkey embryos. These results provide important references for clinical application of ABE.