PDF(Pubmed)
Abstract：
Genome engineering has become more accessible thanks to the CRISPR-Cas9 gene-editing system. However, using this technology in synthetic organs called \"organoids\" is still very inefficient. This is due to the delivery methods for the CRISPR-Cas9 machinery, which include electroporation of CRISPR-Cas9 DNA, mRNA, or ribonucleoproteins containing the Cas9-gRNA complex. However, these procedures are quite toxic for the organoids. Here, we describe the use of the \"nanoblade (NB)\" technology, which outperformed by far gene-editing levels achieved to date for murine- and human tissue-derived organoids. We reached up to 75% of reporter gene knockout in organoids after treatment with NBs. Indeed, high-level NB-mediated knockout for the androgen receptor encoding gene and the cystic fibrosis transmembrane conductance regulator gene was achieved with single gRNA or dual gRNA containing NBs in murine prostate and colon organoids. Likewise, NBs achieved 20%-50% gene editing in human organoids. Most importantly, in contrast to other gene-editing methods, this was obtained without toxicity for the organoids. Only 4 weeks are required to obtain stable gene knockout in organoids and NBs simplify and allow rapid genome editing in organoids with little to no side effects including unwanted insertion/deletions in off-target sites thanks to transient Cas9/RNP expression.
摘要：
由于CRISPR-Cas9基因编辑系统，基因组工程变得更容易获得。然而,在被称为“类器官”的合成器官中使用这项技术仍然非常低效。这是由于CRISPR-Cas9机器的交付方法，其中包括CRISPR-Cas9DNA的电穿孔，mRNA或含有Cas9-gRNA复合物的核糖核蛋白。然而,这些程序对类器官毒性很大。这里,我们描述了“nanoblade（NB）”技术的使用，迄今为止，鼠类和人类组织来源的类器官的基因编辑水平优于此。在用NBs处理后，我们在类器官中达到了高达75％的报告基因敲除。的确,对于雄激素受体编码基因和囊性纤维化跨膜传导调节基因的高水平NB-介导敲除是通过在鼠前列腺和结肠类器官中含有NBs的单gRNA或双gRNA实现的。同样，NBs在人类类器官中实现了20%-50%的基因编辑。最重要的是,与其他基因编辑方法相比，这是对类器官无毒性获得的。仅需要4周才能在类器官中获得稳定的基因敲除,并且NB简化并允许类器官中的快速基因组编辑,由于瞬时Cas9/RNP表达,几乎没有副作用,包括脱靶位点中的不需要的插入/缺失。
