“Though CRISPR precisely targets a gene of interest, due to NHEJ, its effects can be highly variable,” explains Andre Steinecke, Ph.D., Research Fellow and first author of the publication. “CRISPR can leave cells with either fully nonfunctional genes, weakened genes or sometimes even enhance their function. This isn’t such a problem when removing one that causes a very noticeable effect in cells because you can easily visualize the change and absence of the protein coded by the gene. But some, especially genes in the brain, don’t have strikingly obvious effects or are very difficult to visualize. Our goal was to create a widely applicable strategy, capable of reliably determining the exact genetic cause and correlate it to observed phenotype.”
To validate their strategy, the team at MPFI designed CRISPR technology to target a gene in pyramidal neurons encoding a critical structural protein, called Ankyrin-G (AnkG). Normally, the AnkG protein is confined to a specialized region of the neuron known as the axon initial segment (AIS), which is responsible for generating action potentials. When AnkG is removed, the AIS undergoes a noticeable thickening that can be detected using microscopy. With this characteristic feature, neurons that lack AnkG could be readily distinguished and their exact genotype could then be confirmed. They found that predominately, neurons transfected with their CRISPR probe exhibited a loss of AnkG as well as substantially thickened AIS. But a small portion of neurons transfected with CRISPR still exhibited AnkG levels and AIS thickness comparable with wildtype neurons; demonstrating the varying effects of CRISPR on different cells. To probe and confirm the underlying genetic causes, the team then used laser microdissection to isolate and extract individual neurons whose phenotype had already been characterized. Once extracted, the team sequenced each individual cell separately to determine the genotype. They found that their strategy could reliably and reproducibly link observed phenotype to genotype, where neurons lacking AnkG with thickened axons showed loss-of-function mutations in both copies of the gene whereas neurons with normal levels of AnkG either showed mutations in only one copy (neurons transfected with CRISPR) or normal genotypes (control neurons). The team then confirmed their strategy using two additional genes, MeCP2 and Satb2, finding that their process could once again effectively correlate observed feature to underlying genetics.
“CRISPR/Cas9-based gene targeting holds great promise for systematic understanding of the molecular basis underlying the assembly, function, and dysfunction of neural circuits,” notes Hiroki Taniguchi, Ph.D. “The perfect matching between genotypes determined by our single cell sequencing and those deduced from phenotype evaluation, suggests that our approach is a powerful new method capable of enhancing the reliability and expanding the applications of CRISPR-based techniques.”
This work was supported by the Max Planck Florida Institute for Neuroscience, National Institutes of Health Grant, Uehara Memorial Foundation and NARSAD Young Investigator Grant. The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.
Above illustration: The scientists “reading” the DNA of CRISPR/Cas9-edited neurons serve as a metaphor for the newly devised strategy that allows the linking of phenotype to genotype. This method allows the study of CRISPR-mediated effects in cells while accurately ascertaining the exact DNA changes that caused them.
In vivo single cell genotyping of mouse cortical neurons transfected with CRISPR/Cas9
Andre Steinecke*, Nobuhiro Kurabayashi*, Yasufumi Hayano*, Yugo Ishino, and Hiroki Taniguchi
Cell Reports, 28, 325-331.e4.