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In Vivo Single-Cell Genotyping of Mouse Cortical Neurons Transfected with CRISPR/Cas9.

Abstract：

: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 perform in vivo loss-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 phenotypes in vivo. Here, we report a strategy for single-cell genotyping in CRISPR/Cas-transfected neurons that were phenotypically characterized in 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.

journal_name

Cell Rep

journal_title

Cell reports

authors

Steinecke A,Kurabayashi N,Hayano Y,Ishino Y,Taniguchi H

doi

10.1016/j.celrep.2019.06.038

subject

Has Abstract

pub_date

2019-07-09 00:00:00

pages

325-331.e4

issue

2

issn

2211-1247

pii

S2211-1247(19)30804-6

journal_volume

28

pub_type

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