Haplotype-phased common marmoset embryonic stem cells for genome editing using CRISPR/Cas9

Abstract

Due to anatomical and physiological similarities to humans, the common marmoset (Callithrix jacchus) is an ideal organism for the study 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. The genome characterization of these cell lines greatly reinforces these transgenic efforts. It also provides the genomic contexts required for the accurate interpretation of functional genomics data. We performed haplotype-resolved whole-genome characterization 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.

<p>Hepatic Differentiation of Marmoset Embryonic Stem Cells and Functional Characterization of ESC-Derived Hepatocyte-Like Cells</p>

“…The common marmoset (C. jacchus) embryonic stem cell line cj367 11,12 established at the Wisconsin National Primate Research Center (kindly provided by Dr. T. G. Golos) was cultured in E8 medium (Cat# A15169-01; Thermo Fisher Scientific, Waltham, MA), with E8 supplement (1X; Cat# A15171-01; Thermo Fisher Scientific); GlutaMAX (1X; Cat# 35050-061; Life Technologies, Grand Island, NY); lipid concentrate (1:100; Cat# 11905-031; Life Technologies); recombinant human nodal (100 ng/mL; Cat# 3218-ND; R&D Systems, Minneapolis, MN); and glutathione (1.94 μg/mL; Cat# G4251; Sigma Aldrich, St. Louis, MO). Cells were grown on matrigel-coated plates (0.083 μg/ well; Cat# 354230; BD Biosciences, San Jose, CA) and passaged using Accutase (Cat# 7920; Stem Cell Technologies, Vancouver, Canada) when reaching 80% confluency.…”

Utility of Common Marmoset (Callithrix jacchus) Embryonic Stem Cells in Liver Disease Modeling, Tissue Engineering and Drug Metabolism

“…The first set of eight common marmoset embryo–derived pluripotent stem cell lines were isolated in 1996 [ 38 ]. Subsequently, other research groups also established C. jacchus ESC (cjESC) cell lines [ 39 , 40 , 41 , 42 ]. Studies have shown that they can be propagated in vitro both on feeder layers and in feeder-independent culture conditions [ 43 , 44 ], and that they can be genetically modified using CRISPR/Cas9 gene editing and the PiggyBac transposase system [ 45 , 46 ].…”