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使用修改的细胞特异CRISPR方法剖析昼夜基因的神经元特异性功能

Shlesha Richhariya¹, Daniel Shin¹, Jasmine Quynh Le¹

¹HHMI, Brandeis University, Waltham, MA 02454.

Proceedings of the National Academy of Sciences of the United States of America

|July 10, 2023

概括

克里斯普尔基因编辑有效地操纵特定成年果神经元中的基因功能. 这种强大的方法精确地准昼夜钟基因,揭示了对行为节律的新见解.

科学领域:

神经科学是一个神经科学.
遗传学是一个遗传学.
时间生物学时间生物学

背景情况:

在Drosophila melanogaster中,循环节律是由大约75对脑神经元控制的,每个神经元都有独特的功能和基因表达特征.
了解这些独特的分子程序需要精确的,神经元特定的基因操纵技术.
目前的RNAi方法通常不足以用于细胞特异性基因表达操纵,特别是在小的神经元群体中.

研究的目的:

探索和验证一种基于CRISPR的神经元特异的基因编辑策略,用于Drosophila中的时钟基因变异.
研究特定时钟基因 (vrille,Cryptochrome,Pdf) 在调节昼夜行为中的作用.
评估使用可诱导的Cas9和auxin可诱导系统在成年神经元中进行时间基因操纵的疗效.

主要方法:

利用一种基于神经元特异的CRISPR系统,对Drosophila中的时钟基因vrille,Cryptochrome (cry) 和色素分散因子 (Pdf) 进行变异.
在成年神经元中使用可诱导的Cas9和辅酶可诱导的基因表达系统来调节时间基因.
分析了由此产生的表型,以确认基因功能,并确定负责特定光介导行为的神经元子集.

主要成果:

克里斯普尔策略成功地复制了Vrille,cry和Pdf突变的已知表型.
基于CRISPR的基因编辑精确地将Cryptochrome在光介导行为中的功能映射到不同的昼夜神经元子集.
可诱导的Cas9和可诱导的auxin系统都证明了成功地对成年人进行了Pdf的特定淘汰,并重复了功能丧失的表型.

结论:

基于CRISPR的基因编辑是一种高效,可靠和多功能方法,可以暂时操纵特定成年神经元中的基因功能.
这种方法在研究复杂神经回路中的基因功能方面比传统的RNAi具有显著的优势.
这些发现为剖析昼夜节律和其他神经元过程的遗传基础提供了强大的工具.

关键词:

成人特异性的CRISPR是一种成年特异性的CRISPR 细胞类型特定的CRISPR.昼夜活动时间 (Circadian)时钟神经元时钟神经元这就是加密染色.

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