Faculty Collaboration Database

The application of CRISPR technology to high content screening in primary neurons. Mol Cell Neurosci 2017 Apr;80:170-179

Date

01/23/2017

Pubmed ID

28110021

Pubmed Central ID

PMC5393931

DOI

10.1016/j.mcn.2017.01.003

Scopus ID

2-s2.0-85011266012 (requires institutional sign-in at Scopus site)   11 Citations

Abstract

Axon growth is coordinated by multiple interacting proteins that remain incompletely characterized. High content screening (HCS), in which manipulation of candidate genes is combined with rapid image analysis of phenotypic effects, has emerged as a powerful technique to identify key regulators of axon outgrowth. Here we explore the utility of a genome editing approach referred to as CRISPR (Clustered Regularly Interspersed Palindromic Repeats) for knockout screening in primary neurons. In the CRISPR approach a DNA-cleaving Cas enzyme is guided to genomic target sequences by user-created guide RNA (sgRNA), where it initiates a double-stranded break that ultimately results in frameshift mutation and loss of protein production. Using electroporation of plasmid DNA that co-expresses Cas9 enzyme and sgRNA, we first verified the ability of CRISPR targeting to achieve protein-level knockdown in cultured postnatal cortical neurons. Targeted proteins included NeuN (RbFox3) and PTEN, a well-studied regulator of axon growth. Effective knockdown lagged at least four days behind transfection, but targeted proteins were eventually undetectable by immunohistochemistry in >80% of transfected cells. Consistent with this, anti-PTEN sgRNA produced no changes in neurite outgrowth when assessed three days post-transfection. When week-long cultures were replated, however, PTEN knockdown consistently increased neurite lengths. These CRISPR-mediated PTEN effects were achieved using multi-well transfection and automated phenotypic analysis, indicating the suitability of PTEN as a positive control for future CRISPR-based screening efforts. Combined, these data establish an example of CRISPR-mediated protein knockdown in primary cortical neurons and its compatibility with HCS workflows.

Author List

Callif BL, Maunze B, Krueger NL, Simpson MT, Blackmore MG

Author

Murray Blackmore PhD Assistant Professor in the School of Allied Health department at Marquette University

MESH terms used to index this publication - Major topics in bold

Animals
Animals, Newborn
Cells, Cultured
Cerebral Cortex
Clustered Regularly Interspaced Short Palindromic Repeats
Green Fluorescent Proteins
Nerve Tissue Proteins
Neuronal Outgrowth
Neurons
PTEN Phosphohydrolase
RNA, Small Interfering
Rats
Rats, Sprague-Dawley
Transfection
Tubulin