Haplotype-phased Callithrix jacchus embryonic stem cell line for genome editing using CRISPR/Cas9

Due to anatomical and physiological similarities to humans, the common marmoset (Callithrix jacchus), a non-human primate, is an ideal organism for the study of many human diseases. Researchers are currently leveraging genome-editing technologies such as CRISPR/Cas9 to genetically engineer marmosets for the in vivo biomedical modeling of human neuropsychiatric and neurodegenerative diseases. Several marmoset embryonic stem cell (ESC) lines and induced pluripotent stem cell (iPSC) lines established for these purposes are now available and being distributed to various research institutions. The genome characterization of these cell lines can greatly reinforce these transgenic efforts and serve as a valuable resource in many ways. For example, knowing the genomic sequence variants and haplotype phasing information for a particular marmoset cell line allows for the better control of off-target effects in CRISPR/Cas9 experiments and also the ability to make allele-specific edits. It also provides the genomic contexts required for the accurate interpretation of functional genomics data. Here, we performed whole-genome characterization in a haplotype-resolved manner for marmoset ESC line cj367 from the Wisconsin National Primate Research Center. This is the first haplotype-resolved analysis of a marmoset genome and the first whole-genome characterization of any marmoset ESC line. We identified and phased single-nucleotide variants (SNVs) and Indels across the genome. By leveraging this haplotype information, we then compiled a list of cj367 ESC allele-specific CRISPR targeting sites. Furthermore, we demonstrated successful Cas9 Endonuclease Dead (dCas9) expression and targeted localization in cj367 as well as sustained pluripotency after dCas9 transfection by teratoma assay. Lastly, we show that these ESCs can be directly induced into functional neurons in a rapid, single-step process. Our study provides a valuable set of genomic resources for primate transgenics in this post-genome era.