Production of a heterozygous exon skipping model of common marmosets using gene-editing technology

Abstract

Nonhuman primates (NHPs), which are closely related to humans, are useful in biomedical research, and an increasing number of NHP disease models have been reported using gene editing. However, many disease-related genes cause perinatal death when manipulated homozygously by gene editing. In addition, NHP resources, which are limited, should be efficiently used. Here, to address these issues, we developed a method of introducing heterozygous genetic modifications into common marmosets by combining Platinum transcription activator-like effector nuclease (TALEN) and a gene-editing strategy in oocytes. We succeeded in introducing the heterozygous exon 9 deletion mutation in the presenilin 1 gene, which causes familial Alzheimer's disease in humans, using this technology. As a result, we obtained animals with the expected genotypes and confirmed several Alzheimer's disease-related biochemical changes. This study suggests that highly efficient heterozygosity-oriented gene editing is possible using TALEN and oocytes and is an effective method for producing genetically modified animals.

Fig. 1. Validation of candidate TALEN using marmoset embryos.

a, Schematic representation of PSEN1 and TALEN used in the present study. The underlines indicate TALEN binding sites, the blue boxes indicate exons of the marmoset PSEN1 gene, and the red letters (AG) represent the 3′ splice site of intron 8. b, Left: the Surveyor assay of marmoset embryos after TALEN mRNA injection. +N: equal amount of embryo-derived PCR products and WT genomic DNA-derived PCR products were mixed for detecting the biallelic mutations of the PSEN1 gene; +/−: reagent control (the right two columns denote experimental controls included in the Surveyor mutation detection kit); M: GeneRuler 50 bp DNA Ladder (Thermo Fisher Scientific, SM0371). Right: the results of the sequencing analysis of subclones obtained from PCR products. c, Right: evaluation of exon skipping using TALEN mRNA-injected marmoset embryos. Left: illustration of the analysis. The black arrows represent the design positions of the primers used for RT–PCR. Samples with PCR product sizes smaller than 402 bp suggested exclusion of exon 9. M: 100 bp DNA ladder (New England Biolabs; N3231); RT (+): reverse transcriptase-treated samples; RT (−): samples with water added instead of reverse transcriptase. Source data

Fig. 2. PSEN1-modified animals and genotypic analyses.

a, Schematic representation of animal production using marmoset oocytes. b, Photos of obtained PSEN1-ΔE9 marmosets. c, Left: an example of RNA obtained from hair roots of PSEN1-ΔE9 marmoset analyzed by RT–PCR. HR: hair root genome; M: 100 bp DNA ladder (New England Biolabs; N3231); RT (+): reverse transcriptase-treated samples; RT (−): samples with water added instead of reverse transcriptase. Right: results of sequencing analysis of amplified products by RT–PCR obtained from seven mutant animals. Source data

Fig. 3. Biochemical analyses of PSEN1-ΔE9 marmosets.

a, Biochemical analysis of PSEN1ΔE9 marmoset fibroblasts. Western blot analysis of membrane fractions obtained from four WT and two mutant (ΔE9) marmoset fibroblasts using antibodies to NTFs (left) and to CTFs (right) of PSEN1. The arrowheads indicate NTF and CTF produced by endoproteolysis of PSEN1; arrows indicate uncleaved, full-length PSEN1 protein. Na–K ATPase was used as a loading control of the membrane fraction. b, Aβ₄₀ and Aβ₄₂ in the plasma of 13 WT and 8 PSEN1-ΔE9 marmosets were quantified using Simoa. Aβ₄₃ levels were below the detection limit. The data represent mean ± standard error of the mean (WT: n = 17, PSEN1-ΔE9: n = 8). Statistical analyses were performed using the Student’s two-tailed t-test for the Aβ₄₀ and Aβ₄₂ concentration (**P < 0.01, ****P < 0.0001), and Mann–Whitney U test for the Aβ₄₂/Aβ₄₀ ratio (****P < 0.0001). Source data