Robust and efficient knock-in in embryonic stem cells and early-stage embryos of the common marmoset using the CRISPR-Cas9 system

Mari Nakamura,Erika Sasaki,Noriyuki Kishi,Yuta Takeda,Seiji Shiozawa,Sho Yoshimatsu,Takefumi Sone,Hideyuki Okano,Junko Okahara

doi:10.1038/s41598-018-37990-w

Abstract

Genome editing technology greatly facilitates the genetic modification of various cells and animals. The common marmoset (Callithrix jacchus), a small non-human primate which exhibits high reproductive efficiency, is a widely used animal model in biomedical research. Developing genome editing techniques in the common marmoset will further enhance its utility. Here, we report the successful establishment of a knock-in (KI) method for marmoset embryonic stem cells (ESCs), which is based on the CRISPR-Cas9 system. The use of CRISPR-Cas9, mediated by homologous recombination (HR), enhanced the KI efficiency in marmoset ESCs. Furthermore, we succeeded in performing KI in early-stage marmoset embryos. In the course of the experiments, we found that HR in the marmoset ESCs is innately highly efficient. This suggested that the marmoset possesses a repair mechanism for DNA double-strand breaks. The current study will facilitate the generation of genetically modified marmosets and gene function analysis in the marmoset.

Highlights

There are two major pathways for repairing DNA double-strand breaks (DSBs)[1,2,3]
Cas[9] system works in marmoset cells, we introduced marmoset-specific gRNA sequences into pSpCas9-2A-Puro (Cas9-gRNA vector; PX459) and evaluated the genomic cleavage activity (GCA) of Cas[9] and the gRNAs in cjESCs by transfecting each Cas9-gRNA vector and transiently selecting the transfected cjESCs with puromycin
We demonstrated the impact of genome editing through targeting of the ACTB gene with a promoter-trap strategy and found that clustered regularly interspaced palindromic repeats (CRISPR)-Cas[9] increased the number of homologous recombinants

Summary

Results

Evaluation of KI efficiency in cjESCs using the CRISPR-Cas[9] system. Cas[9] system works in marmoset cells, we introduced marmoset-specific gRNA sequences (summarized in Supplementary Table 2) into pSpCas9-2A-Puro (Cas9-gRNA vector; PX459) and evaluated the genomic cleavage activity (GCA) of Cas[9] and the gRNAs in cjESCs by transfecting each Cas9-gRNA vector and transiently selecting the transfected cjESCs with puromycin (see Experimental procedures). To demonstrate the utility of the PLP1-EGFP KI reporter, we differentiated one homozygous-KI cjESC clone (Cas9− #11) into neuronal cells including OLs following excising the loxP-flanked PGK-Neo cassette (Supplementary Fig. S5a–e). In order to verify that a high ratio of KI clones was obtained from G418-resistant Cas9− cjESC clones (Fig. 2e), we performed genotyping PCR using bulk genomic DNA extracted from the G418-resistant colonies from each well which was transfected with PLP1-EGFP TV, either with or without the Cas9-gRNA (gRNA: PLP1-2; Cas9(+) or Cas9(−) wells). KI clones generated by transfecting cjESCs with the TV and Cas9-gRNA targeting exon 8 (FOXP2-4) had humanized sequences in exon 8 in all of the KI alleles (Fig. 3e, top, and Supplementary Table 5). By using the microinjection and electroporation conditions we developed and optimized, we succeeded in efficiently introducing gene modifications in marmoset embryos, resulting in either KI or KO

Discussion

Author Contributions

Additional Information