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

Published on Dec 1, 2019in Scientific Reports4.01
· DOI :10.1038/s41598-019-49110-3
Wakako Kumita2
Estimated H-index: 2
(Central Institute for Experimental Animals),
Kenya Sato3
Estimated H-index: 3
(Central Institute for Experimental Animals)
+ 10 AuthorsErika Sasaki21
Estimated H-index: 21
(Central Institute for Experimental Animals)
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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.
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