Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA.
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA.
Neuron. 2019 Oct 23;104(2):227-238.e7. doi: 10.1016/j.neuron.2019.07.005. Epub 2019 Aug 5.
Gaining independent genetic access to discrete cell types is critical to interrogate their biological functions as well as to deliver precise gene therapy. Transcriptomics has allowed us to profile cell populations with extraordinary precision, revealing that cell types are typically defined by a unique combination of genetic markers. Given the lack of adequate tools to target cell types based on multiple markers, most cell types remain inaccessible to genetic manipulation. Here we present CaSSA, a platform to create unlimited genetic switches based on CRISPR/Cas9 (Ca) and the DNA repair mechanism known as single-strand annealing (SSA). CaSSA allows engineering of independent genetic switches, each responding to a specific gRNA. Expressing multiple gRNAs in specific patterns enables multiplex cell-type-specific manipulations and combinatorial genetic targeting. CaSSA is a new genetic tool that conceptually works as an unlimited number of recombinases and will facilitate genetic access to cell types in diverse organisms.
获得独立的遗传方法来获取离散的细胞类型对于研究它们的生物学功能以及进行精确的基因治疗至关重要。转录组学使我们能够以极高的精度对细胞群体进行分析，揭示了细胞类型通常由独特的遗传标志物组合来定义。鉴于缺乏基于多个标志物来靶向细胞类型的适当工具，大多数细胞类型仍然无法进行遗传操作。在这里，我们介绍了 CaSSA，这是一个基于 CRISPR/Cas9（Ca）和称为单链退火（SSA）的 DNA 修复机制的平台，用于创建无限数量的遗传开关。CaSSA 允许独立的遗传开关的工程设计，每个开关都响应特定的 gRNA。以特定模式表达多个 gRNA 可实现多重细胞类型特异性操作和组合遗传靶向。CaSSA 是一种新型的遗传工具，在概念上可以作为无限数量的重组酶发挥作用，将有助于在不同生物体中获得细胞类型的遗传方法。
