Homologous recombination-mediated targeted integration in monkey embryos using TALE nucleases

Abstract

Background: Non-human primate (NHP) models can closely mimic human physiological functions and are therefore highly valuable in biomedical research. Genome editing is now developing rapidly due to the precision and efficiency offered by engineered site-specific endonuclease-based systems, such as transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) system. It has been demonstrated that these programmable nucleases can introduce genetic changes in embryos from many species including NHPs. In 2014, we reported the first genetic editing of macaques using TALENs and CRISPR/Cas9. Subsequently, we characterized the phenotype of a methyl CpG binding protein 2 (MECP2)-mutant cynomolgus monkey model of Rett syndrome generated using the TALEN approach. These efforts not only accelerated the advance of modeling genetic diseases in NHPs, but also encouraged us to develop specific gene knock-in monkeys. In this study, we assess the possibility of homologous recombination (HR)-mediated gene replacement using TALENs in monkeys, and generate preimplantation embryos carrying an EmGFP fluorescent reporter constructed in the OCT4 gene.

Result: We assembled a pair of TALENs specific to the first exon of the OCT4 gene and constructed a donor vector consisting of the homology arms cloned from the monkey genome DNA, flanking an EmGFP cassette. Next, we co-injected the TALENs-coding plasmid and donor plasmid into the cytoplasm of 122 zygotes 6-8 h after fertilization. Sequencing and immunofluorescence revealed that the OCT4-EmGFP knock-in allele had been successfully generated by TALENs-mediated HR at an efficiency of 11.3% (7 out of 62) or 11.1% (1 out of 9), respectively, in monkey embryos.

Conclusion: We have successfully, for the first time, obtained OCT4-EmGFP knock-in monkey embryos via HR mediated by TALENs. Our results suggest that gene targeting through TALEN-assisted HR is a useful approach to introduce precise genetic modification in NHPs.

Keywords: Gene editing; Homologous recombination; Non-human primate; TALENs.

Fig. 1

Workflow of TALEN-mediated generation of a monkey embryo carrying an EmGFP reporter in the OCT4 gene. TALENs-coding plasmids, pTALEN-Maca-oct4-E1-F/R, and the donor vector Donor-E1-PKID-EmGFP that targets exon 1 of the OCT4 gene were designed and co-injected into the cytoplasm of a zygote 6–8 h after fertilization. Treated embryos at the blastocyst, morula and 16-cell stages were collected and analyzed

Fig. 2

Schematic overview of the OCT4-EI-TALEN construction and the SSA assay for testing the OCT4-TALEN-coding plasmids. a The TALEN-targeted sequences within exon 1 of the OCT4 gene are labeled in red. Assembled Repeat Variable Diresidue (RVD) repeats are represented schematically as boxes and labeled in yellow, red, blue and green. b The overview of the GFP-reporter system for SSA. Red lines represent 541 bp oligonucleotides near the TALEN-targeted sites amplified from genomic DNA. c Fluorescence intensity of GFP in 293 T cells 19 h or 49 h post transfection with OCT4-E1-TALEN-coding plasmids and the reporter vector pJL4-SSA (left) or controls transfected only with the reporter plasmid (right)

Fig. 3

Detection of TALEN-induced mutations in the genomes of monkey embryos. a The T7EN1 cleavage assay for on-target efficiency of TALENs .The positive cleavage bands were visible in 7 samples in addition to 0106.M2. b Sequencing results showed different indels at the target site. The two short base sequences in red are the target sites for the TALEN pair, while the 17 bp between them comprise the region cleaved by FokI

Fig. 4

Genotyping and immunofluorescence of embryos generated by TALEN-mediated genome engineering. a Schematic overview of the strategy to generate an OCT4-EmGFP knock-in allele. The homologous arms of the donor vector are indicated as LA (839 bp) and RA (1001 bp). PCR primers used for PCR genotyping are shown as arrows in different colors. b PCR products obtained using primers L1-F and L1-R were sequenced. Sequence across the targeted region confirmed the correct fusion of EmGFP to the first exon of OCT4. c EmGFP: PCR genotyping using primers G-F and G-R produced bands of 421-bp fragment in the samples from nine embryos, suggesting the possible insertion or precise knock-in of the EmGFP gene. L1: PCR genotyping using primers L1-F and L1-R produced large products (3259 bp) in seven embryo samples, indicating the EmGFP sequence was integrated. The samples 0806.16C1, 0308 M4 and 0308.B1 only contain the larger product, suggesting either both alleles were targeted, or one allele failed to amplify. S1 and S2: PCR genotyping using primers S1-F and S1-R, S2-F and S2-R produced bands with correct size (1795 bp and 2109 bp) in the samples from the targeted embryos. d Immunostaining of targeted blastocysts using anti-GFP antibody showed a signal in the ICM. Scale bar, 50 μM