Generation of a Tyrosine hydroxylase (TH)-2A-Cre knock-in non-human primate model by homology directed repair-biased CRISPR-Cas9 genome editing.
Non-human primates (NHPs) are the most closed animal model to humans, thus gene engineering technology in these species should hold great promise for the elucidation of higher brain functions and human disease modeling. Knock-in (KI) gene targeting is a versatile approach to modify gene(s) of interest, but it generally suffers from the low efficiency of homology-directed repair (HDR) in mammalian cells, especially in non-expressed gene loci. In the current study, we generated a Tyrosine hydroxyrase (TH)-2A-Cre knock-in model of the common marmoset monkey (marmoset; Callithrix jacchus) by an HDR-biased CRISPR-Cas9 genome editing approach using Cas9-DN1S and RAD51. Furthermore, using marmoset embryonic stem cells (ESCs), we validated the functionality of the knock-in Cre reporter allele in vitro. Collectively, the current study paves the invaluable way for versatile gene engineering in NHPs, which may make a significant step for further biomedical and preclinical applications.