In Vivo Single-Cell Genotyping of Mouse Cortical Neurons Transfected with CRISPR/Cas9.
In Vivo Single-Cell Genotyping of Mouse Cortical Neurons Transfected with CRISPR/Cas9.
复制标题
转染 CRISPR/Cas9 的小鼠皮质神经元的体内单细胞基因分型。
DOI：
10.1016/j.celrep.2019.06.038
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发表时间：
2019
期刊：
影响因子：
8.8
通讯作者：
Taniguchi,Hiroki
中科院分区：
文献类型：
--
作者：
Steinecke,André;Kurabayashi,Nobuhiro;Hayano,Yasufumi;Ishino,Yugo;Taniguchi,Hiroki
基于 CRISPR/Cas 的技术彻底改变了解决各种神经生物学问题的遗传学方法。 CRISPR/Cas 将突变引入目标基因的能力使我们能够在不产生基因工程小鼠的情况下进行体内功能丧失实验。然而，由于缺乏可靠的方法来确定单个 CRISPR/Cas 转染细胞的基因型，因此无法明确识别其体内表型的遗传原因。在这里，我们报告了一种对 CRISPR/Cas 转染的神经元进行单细胞基因分型的策略，这些神经元在体内进行了表型表征。我们证明，皮层切片的重新切片和随后的激光显微切割使我们能够分离单个 CRISPR/Cas 转染的神经元。对单个参考神经元中含有 CRISPR/Cas 靶向基因组区域的 PCR 产物进行测序，得到的基因型与从其靶蛋白表达和表型推断的基因型完全一致。因此，我们的研究建立了一个强大的策略来确定 CRISPR/Cas 转染神经元中基因型和表型之间的因果关系。
CRISPR/Cas-based technologies have revolutionized genetic approaches to addressing a wide range of neurobiological questions. The ability of CRISPR/Cas to introduce mutations into target genes allows us to performin vivoloss-of-function experiments without generating genetically engineered mice. However, the lack of a reliable method to determine genotypes of individual CRISPR/Cas-transfected cells has made it impossible to unambiguously identify the genetic cause of their phenotypesin vivo. Here, we report a strategy for single-cell genotyping in CRISPR/Cas-transfected neurons that were phenotypically characterizedin vivo. We show that re-sectioning of cortical slices and subsequent laser microdissection allow us to isolate individual CRISPR/Cas-transfected neurons. Sequencing of PCR products containing a CRISPR/Cas-targeted genomic region in single reference neurons provided genotypes that completely correspond with those deduced from their target protein expression and phenotypes. Thus, our study establishes a powerful strategy to determine the causality between genotypes and phenotypes in CRISPR/Cas-transfected neurons.