Generation of knock-in cynomolgus monkey via CRISPR/Cas9 editing

Genetically modified monkey models that exhibit human disease symptoms are versatile tools for studying human disease mechanisms and advancing therapeutic treatments¹. However, generation of gene-targeted monkeys remains very challenging, due to the long breeding cycle and low gene-editing efficiency^1,2. The CRISPR/Cas9 system has been used to generate gene-targeted animals in many species by directly injecting Cas9 mRNA and single-guide RNAs (sgRNAs) into pronuclear stage zygotes³. Several recent studies have shown that the CRISPR/Cas9 system could also be used to generate gene-knockout monkeys^{4,5,6,7,8,9,10}. However, no gene knock-in monkey has been reported, presumably because of the low knock-in rate².

We previously reported that homology-mediated end joining (HMEJ)-based method could efficiently achieve targeted integration in the embryos of cynomolgus monkeys¹¹. However, the low blastocyst rate of the injected embryos (26.7%, 4 out of 15) may hinder the generation of live-birth monkeys¹¹. We thus further optimized the zygote injection conditions, including the concentration of donor plasmids and the total injection volume, to improve the knock-in efficiency while maintaining the normal embryonic development. In this study, we aimed to insert Actb (intron 4 to exon 5)-p2A-mCherry into the intron 4 of the Actb gene (highly expressed in most cells/tissues) to achieve mCherry expression under the control of the Actb promoter (Supplementary information, Figure S1A). The knock-in efficiencies were evaluated by mCherry fluorescence signals in blastocysts (Supplementary information, Figure S1B). By co-injection of different volumes (∼4-10 pl) and concentrations of donor plasmid (50-100 ng/μl) together with Cas9 mRNA (100 ng/μl) and sgRNA (50 ng/μl) into fertilized monkey oocytes, we found that ∼4 pl of total volume and 100 ng/μl of donor plasmid could achieve both high knock-in efficiency (5 out of 6) and high blastocyst rate (6 out of 11; Supplementary information, Figure S1C and S1D).

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