Efficient generation of Knock-in/Knock-out marmoset embryo via CRISPR/Cas9 gene editing

Wakako Kumita,Erika Sasaki,Yang Zhou,Takeshi Harada,Kengo Sato,Yoko Kurotaki,Hideyuki Okano,Guoping Feng,Atsu Aiba,Yasuhiro Suzuki,Yasubumi Sakakibara,Kenya Sato,Noriyuki Kishi

doi:10.1038/s41598-019-49110-3

Abstract

Genetically modified nonhuman primates (NHP) are useful models for biomedical research. Gene editing technologies have enabled production of target-gene knock-out (KO) NHP models. Target-gene-KO/knock-in (KI) efficiency of CRISPR/Cas9 has not been extensively investigated in marmosets. In this study, optimum conditions for target gene modification efficacies of CRISPR/mRNA and CRISPR/nuclease in marmoset embryos were examined. CRISPR/nuclease was more effective than CRISPR/mRNA in avoiding mosaic genetic alteration. Furthermore, optimal conditions to generate KI marmoset embryos were investigated using CRISPR/Cas9 and 2 different lengths (36 nt and 100 nt) each of a sense or anti-sense single-strand oligonucleotide (ssODN). KIs were observed when CRISPR/nuclease and 36 nt sense or anti-sense ssODNs were injected into embryos. All embryos exhibited mosaic mutations with KI and KO, or imprecise KI, of c-kit. Although further improvement of KI strategies is required, these results indicated that CRISPR/Cas9 may be utilized to produce KO/KI marmosets via gene editing.

Highlights

Several types of gene editing technologies including zinc finger nucleases (ZFNs)[8], transcription activator-like effector nucleases (TALENs)[9] and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)[10,11] have been developed
Target gene modifications in blastomeres were analyzed to verify mosaicism in embryos following CRISPR/mRNA or CRISPR/nuclease injection in order to predict whether first generation target gene KO/KI animals may display objective phenotypes
CRISPR/nuclease showed 100% modification in marmoset whole embryos for both target genes, modification rates determined via CEL-1 assay were not significantly different compared to those for CRISPR/mRNA due to both artificial nuclease injections exhibiting a high degree of efficiency in target gene modification (Table 1)

Summary

Introduction

Several types of gene editing technologies including zinc finger nucleases (ZFNs)[8], transcription activator-like effector nucleases (TALENs)[9] and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)[10,11] have been developed. All these technologies involve artificial nucleases and induce double-strand breaks (DSB) in a specific gene of the target genome. DSB repair may lead to non-homologous end-joining causing a frameshift, which disrupts the function of the target gene Such gene alteration techniques can be applied to preimplantation embryos, making it possible to generate target-gene KO/KI models in many animal species including NHPs that lack chimeric competent ESCs12–14. Gene modification activity and mosaicism frequency of CRISPR/Cas[9] related to c-kit and Shank[3] and their mosaic mutation rates in marmoset embryos were investigated

Methods

Results

Conclusion