Savell Katherine E, Sultan Faraz A, Day Jeremy J
Department of Neurobiology, University of Alabama at Birmingham, Birmingham, AL, USA.
Behavioral Neuroscience Research Branch, National Institute on Drug Abuse Intramural Research Program, Baltimore, Maryland, USA.
Bio Protoc. 2019 Sep 5;9(17):e3348. doi: 10.21769/BioProtoc.3348.
Robust and efficient gene expression control enables the study of a gene's function in the central nervous system. Advances in CRISPR-based technology provide new avenues not only for gene editing, but for complex transcriptional control. Here, we describe a protocol to generate high-titer lentiviruses with neuron-optimized CRISPR-activation constructs (dual lentiviruses consisting of a gene-specific single guide RNA and the CRISPR-activator) for use in primary neurons or in the adult brain . This protocol enables modular, scalable, and multiplexable gene regulation in the nervous system and does not require a transgenic model organism.
强大而高效的基因表达控制有助于研究基因在中枢神经系统中的功能。基于CRISPR的技术进展不仅为基因编辑，也为复杂的转录控制提供了新途径。在此，我们描述了一种方案，用于生成具有神经元优化的CRISPR激活构建体（由基因特异性单向导RNA和CRISPR激活剂组成的双慢病毒）的高滴度慢病毒，以用于原代神经元或成年大脑。该方案能够在神经系统中实现模块化、可扩展和多重基因调控，并且不需要转基因模式生物。
