向导 RNA 的顺序激活以实现连续的 CRISPR-Cas9 活性。
Sequential Activation of Guide RNAs to Enable Successive CRISPR-Cas9 Activities.
机构信息
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, USA.
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, USA.
出版信息
Mol Cell. 2021 Jan 21;81(2):226-238.e5. doi: 10.1016/j.molcel.2020.12.003. Epub 2020 Dec 29.
Currently, either highly multiplexed genetic manipulations can be delivered to mammalian cells all at once or extensive engineering of gene regulatory sequences can be used to conditionally activate a few manipulations. Here, we provide proof of principle for a new system enabling multiple genetic manipulations to be executed as a preprogrammed cascade of events. The system leverages the programmability of the S. pyogenes Cas9 and is based on flexible arrangements of individual modules of activity. The basic module consists of an inactive single-guide RNA (sgRNA)-like component that is converted to an active state through the effects of another sgRNA. Modules can be arranged to bring about an algorithmic program of sequential genetic manipulations without the need for engineering cell-type-specific promoters or gene regulatory sequences. With the expanding diversity of available tools that use spCas9, this sgRNA-based system provides multiple levels of interfacing with mammalian cell biology.
目前，要么可以一次性将高度多重的遗传操作递送到哺乳动物细胞中，要么可以对基因调控序列进行广泛的工程改造，以有条件地激活少数操作。在这里，我们提供了一个新系统的原理证明，该系统允许将多个遗传操作作为预定的级联事件来执行。该系统利用了 S. pyogenes Cas9 的可编程性，并且基于单个活性模块的灵活排列。基本模块由一个不活跃的单指导 RNA（sgRNA）样组件组成，该组件通过另一个 sgRNA 的作用转换为活跃状态。可以对模块进行排列，以实现顺序遗传操作的算法程序，而无需工程改造细胞类型特异性启动子或基因调控序列。随着使用 spCas9 的可用工具的多样性不断扩大，这种基于 sgRNA 的系统为与哺乳动物细胞生物学的多种接口提供了支持。
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