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

Cell Rep. 2019 Jul 9;28(2):325-331.e4. doi: 10.1016/j.celrep.2019.06.038.

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.

Keywords: Ankyrin-G; CRISPR/Cas9; MeCP2; Satb2; gene knockout; laser microdissection; single-cell genotyping.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics*
  • Genotype
  • Mice
  • Neurons / metabolism*
  • Transfection