Generation of a tyrosine hydroxylase-2A-Cre knockin non-human primate model by homology-directed-repair-biased CRISPR genome editing

Non-human primates (NHPs) are the closest animal model to humans; thus, gene engineering technology in these species holds great promise for the elucidation of higher brain functions and human disease models. Knockin (KI) gene targeting is a versatile approach to modify gene(s) of interest; however, 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 hydroxylase (TH)-2A-Cre KI model of the common marmoset monkey (marmoset; Callithrix jacchus) using an HDR-biased CRISPR-Cas9 genome editing approach using Cas9-DN1S and RAD51. This model should enable labeling and modification of a specific neuronal lineage using the Cre-loxP system. Collectively, the current study paves the way for versatile gene engineering in NHPs, which may be a significant step toward further biomedical and preclinical applications. [Display omitted] •We generate a TH-2A-Cre knockin marmoset model•Cas9-DN1S and Rad51 proteins facilitate knockin in marmoset zygotes•Introduced Cre expression is validated through reprogramming•Introduced Cre activity is validated in an in vitro model Gene engineering in the common marmoset has the potential to provide unique insights into human disease and brain function because of its similarity to humans. To expand the utility of the model system, we attempted and succeeded in generating a gene knockin marmoset using CRISPR-Cas9-mediated and HR-based genome editing. By targeting TH, a specific marker for dopaminergic, adrenergic, and noradrenergic neurons, we produced a highly faithful TH reporter system in the marmoset via genome editing. Yoshimatsu et al. generate a TH-2A-Cre knockin marmoset model. Their approaches include validating the knockin construct in embryonic stem cells, injecting Cas9-DN1S and Rad51 proteins with crRNA/tracrRNA to enhance knockin efficiency, genotyping embryos thorough whole-genome amplification, and reprogramming somatic fibroblasts for validation of the knocked-in reporter.