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Research Progress

CRISPR/Cas9-Mediated HMEJ-Based Strategy to Generate Knock-In Cynomolgus Monkey

Jan 15, 2018

Genetically modified monkey models that exhibit human disease symptoms are versatile tools to study human disease mechanisms and advance therapeutic treatments. Due to the long breeding cycle (5-6 years), small litter size (1 per birth) and low gene-editing efficiency, the generation of gene-targeted monkeys remains a challenge.

Several recent studies have shown that the CRISPR/Cas9 system could be used to generate gene-knockout monkeys. However, no successful gene knock-in monkey has been reported to date, presumably because of the low knock-in efficiency.

In a recent study published in Cell Research , Dr. YANG Hui’s Lab and Dr. SUN Qiang's group at the Institute of Neuroscience of Chinese Academy of Sciences demonstrated that homology-mediated end joining (HMEJ)-based strategy can generate knock-in cynomolgus monkey using CRISPR/Cas9. They successfully generated targeted knock-in cynomolgus monkey using CRISPR/Cas9-mediated HMEJ-based strategy for the first time in the world.

Based on a previous report, homology-mediated end joining (HMEJ)-based method could efficiently achieve targeted integration in the embryos of cynomolgus monkeys.  The researchers further optimized the zygote injection conditions, including the concentration of donor plasmids and the total injection volume, in order to improve the knock-in efficiency while maintaining the normal embryonic development. 

In this study, they inserted p2A - mCherry into the Actb locus to achieve mCherry expression under the control of the Actb promoter. Successfully delivery at full term yielded a set of twin babies and three female singletons. Direct epifluorescence of the toes of newborn babies showed that two monkeys exhibited visible mCherry fluorescence, as compared to wild-type newborn monkeys. 

Tissue sections obtained from the tail, toe, gut, muscle, heart, kidney, brain and ovary of the two deceased female monkeys showed mosaic expression of mCherry expression. Notably, the researchers detected mCherry expression in most part of the ovaries of monkeys, implying the potential capability of germline transmission of the knock-in monkeys. 

Sequencing of PCR-based genotyping from all samples confirmed the correct transgene integration at 5’ and 3’ junctions. Furthermore, Southern blot analysis confirmed the correct transgene integration and no additional randomly integrated transgene. 

Taken together, the researchers successfully achieved CRISPR/Cas9-initiated transgene targeted integration using HMEJ strategy in monkey genome by one-cell embryo microinjection. With robust DNA knock-in efficiency and high fidelity, the HMEJ-based knock-in strategy opens up the possibility of generating targeted genetic modifications in monkeys.

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