CRISPR/Cas9基因编辑技术在脑科学中的应用策略

Strategy for applying CRISPR/Cas9 gene editing technology in neuroscience

基因编辑是对生物体基因组的目标基因进行精确切割、插入等操作.CRISPR/Cas9技术是基于向导RNA识别DNA靶序列,Cas9蛋白作为核酸酶切割DNA靶点来实现基因编辑.该技术自2012年报道以来已被不断改进,因具有普适、高效、简便等优点,迅速成为现阶段应用最广的基因编辑技术.在脑科学领域,CRISPR/Cas9技术不仅可应用于离体神经细胞,也可以在受精卵期、胚胎期或成年期应用;应用目的涉及脑基因与功能研究、基因敲除/敲入小鼠模型的构建、某些疾病的实验性治疗等.尤其是在一些遗传性疾病如视网膜色素变性、亨廷顿病的动物模型上,CRISPR/Cas9方法已经初步展示了令人鼓舞的治疗效果.未来,该技术将会在精确编辑效率与可控性方面有进一步提升,并可能在脑定向导入方法、脑神经环路解析等专业应用环节获得显著的发展.

The gene editing technology enables cutting or/and inserting the target gene precisely. The CRISPR（clustered regularly interspaced short palindromic repeats）/Cas9（CRISPR-associated protein 9） system relies on two major elements, a guide RNA（gR NA） that recognizes a specific DNA sequence, and a nuclease Cas9 that cuts the target DNA and creates double-strand breaks（DSBs）. Among a few gene-editing technologies available, CRISPR/Cas9 system has showed tremendous advantages over the others, mainly due to high efficiency of Cas9 and simple design of g RNA. As a versatile and powerful tool for genome engineering, the CRISPR/Cas9 technology has been used to generate genetically modified mice with unprecedented simplicity and speed, simply by timed delivery of Cas9/g RNA ribonucleoproteins（RNPs） into pronuclear-stage zygotes. The modified protocol introduces Cas9 protein instead of Cas9 mR NA by rapid electroporation, which enables the gene editing occurring before the first replication of the mouse genome, thus generating non-mosaic mutant embryos. Furthermore, it could be used to produce multiple gene mutations in a single mouse by co-delivering several g RNAs targeted to different genes. Herein, we summarize current applications of CRISPR/Cas9 technology in the field of neuroscience, and aim to provide concise information and perspectives for better utilizing this technology. In brain research, the CRISPR/Cas9 system can be applied either at the one-cell stage of the fertilized eggs in the form of Cas9/g RNA ribonucleoproteins, or at the stage of embryos or adults in the form of plasmid DNA or the viral vectors（commonly AAV variants） respectively. To deliver the system specifically into the developmental or adult brain, in utero electroporation, or stereotaxic injection is commonly employed. Several reports show that AAV-assisted Cas9/g RNA system could achieve a satisfied efficiency for genome editing in the adult mouse brain, but results vary depending on Cas9 activity, g RNA design, detection methods, and the condition of endogenous DNA repair mechanisms. Remarkable efforts have also been made to enhance the incidence of homology-directed repair for precise gene modifications. Regarding therapeutic genome editing, a couple of recent in vivo studies demonstrate that CRISPR/Cas9 system could contract or remove disease-causing alleles in animal models of certain hereditary diseases such as retinitis pigmentosa and Huntington＇s disease, raising hope for translating therapeutic genome editing to clinical patients. In addition, we discuss major challenges and critical improvements for this technology, including a few modifications for promoting precise editing in non-dividing cells in the case of adult brain. In the future, application of CRISPR/Cas9 technology would be enhanced greatly in neuroscience by developing cell type-specific, timed delivery system in vivo and by combining with other powerful techniques for dissecting brain neural circuits in health and disease.