提高CRISPR/Cas9在猴胚胎中基因打靶精确性的研究

CRISPR/Cas9 technology has been widely used in many fields of life sciences, especially as a powerful new tool for genome editing. Its ability to modify genomes in embryos opens up a new avenue to generate genetically modified animal models without the need to use embryonic stem cells. Indeed, CRISPR-Cas9 has been used to target genes in large animals whose embryonic stem cells are not available for genetic manipulation..Unfortunately, however, this technology also creates mosaic mutations in embryos, resulting in different mutations in different types of cells in the same animal. Mosaic mutations can be an obstacle to obtaining genetically modified animals that can faithfully mimic genetic mutations in human patients. More importantly, mosaicism can significantly affect the precision of gene therapy when CRISPR-Cas9 technology is used. Although many studies have tried to reduce the mosaic mutations by using dual gRNAs or direct injection of Cas9 protein to target genes, the mosaic issue remains to be resolved for genetically engineering non-human primate models. .Recently，we have modified Cas9 by shortening its half-life via adding the ubiquitin-proteasome degradation signal (Ubi-GGRGK) to the N-terminus of Cas9 (Ubi-Cas9). We found that this modification decreases Cas9-mediated mosaic mutations in monkey embryos. To completely remove mosaicism, we will tag different degrons to Cas9 to reduce its half-life and identify a modified Cas9 that is able to generate the same DNA mutations in all individual cells in embryos. Achieving this goal is a necessary prerequisite for generating live animals with genetic modification that is identical to the mutation occurring in germline cells in humans. Also, this technology will help the future precision gene therapy.

CRISPR/Cas9技术现已被广泛用来建立基因修饰动物疾病模型。然而，CRISPR/Cas9技术也会产生胚胎嵌合突变，造成同一个动物个体中不同细胞携带不同的基因突变类型。CRISPR/Cas9技术的嵌合效应影响了动物模型建立的有效性和基因修饰的精确性，是制约建立能真正反映病人基因突变类型与病理的大动物模型的一大技术障碍。我们最近发表的文章表明，给Cas9蛋白添加降解信号可降低Cas9蛋白在食蟹猴胚胎细胞中的半衰期，有助于降低Cas9蛋白对猴胚胎基因打靶的嵌合效应。然而，为了达到完全消除CRISPR/Cas9的嵌合效应，有必要对Cas9进行进一步的改造。本项目将通过添加不同降解信号改变Cas9蛋白的半衰期及活性，希望找到一种能够在猴胚胎上建立所有单细胞携带相同基因突变的CRISPR/Cas9基因编辑技术。为更有效建立大动物疾病模型提供可靠的技术支持，同时有助于精准基因治疗的实施。

CRISPR/Cas9技术现已被广泛应用来建立基因修饰动物疾病模型，然而，CRISPR/Cas9技术也会产生胚胎嵌合突变，造成同个动物个体不同类型的细胞携带不同的基因突变类型。CRISPR/ Cas9技术的嵌合效应影响了动物模型建立的有效性和基因修饰的精确性，是制约建立更加有效的非人灵长类动物模型的一大技术障碍。我们最近发表的文章表明，给Cas9蛋白添加降解信号可降低Cas9蛋白在食蟹猴胚胎细胞中的半衰期，助于降低CRISPR/Cas9对猴胚胎的嵌合效应。因此，为了达到更好消除CRISPR/Cas9的嵌合效应，我们进一步改造了Cas9蛋白，并取得了几种不同半衰期的Cas9蛋白载体，通过体内外的验证，这些改造的Cas9蛋白均能够发挥基因编辑作用。通过应用这些改造的Cas9蛋白建立了多种动物模型。同时我们利用CRISPR/Cas9技术，建立了世界首例的亨廷顿基因敲入猪模型，首次将人的htt基因精确插入到猪的基因组中，并成功实现了稳定传代，可更精准模拟病人。为研究人类自闭症，我们建立了Shank3基因敲除猴模型，该模型也能很好的模拟自闭症的主要症状，并且用药后能够取得与患者类似的疗效。这些研究均为神经疾病机制及临床前治疗策略研究奠定基础。

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