Skip to Content
MilliporeSigma
  • The application of CRISPR technology to high content screening in primary neurons.

The application of CRISPR technology to high content screening in primary neurons.

Molecular and cellular neurosciences (2017-01-23)
Ben L Callif, Brian Maunze, Nick L Krueger, Matthew T Simpson, Murray G Blackmore
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-β-Tubulin III antibody produced in rabbit, affinity isolated antibody, buffered aqueous solution
Sigma-Aldrich
Deoxyribonuclease I from bovine pancreas, Type II, lyophilized powder, Protein ≥80 %, ≥2,000 units/mg protein
Sigma-Aldrich
Poly-D-lysine hydrobromide, mol wt 30,000-70,000
Sigma-Aldrich
Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane, 1-2 mg/mL in Tris-buffered saline, 0.2 μm filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
Anti-NeuN Antibody, clone A60, clone A60, Chemicon®, from mouse