title: The application of CRISPR technology to high content screening in primary neurons.
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.
authors.affiliation: Department of Biomedical Sciences, Marquette University, 53201, USA.
authors.forename: Ben L
authors.initials: BL
authors.lastname: Callif
authors.affiliation: Department of Biomedical Sciences, Marquette University, 53201, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Maunze
authors.affiliation: Department of Biomedical Sciences, Marquette University, 53201, USA.
authors.forename: Nick L
authors.initials: NL
authors.lastname: Krueger
authors.affiliation: Department of Biomedical Sciences, Marquette University, 53201, USA.
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Simpson
authors.affiliation: Department of Biomedical Sciences, Marquette University, 53201, USA. Electronic address: murray.blackmore@marquette.edu.
authors.forename: Murray G
authors.initials: MG
authors.lastname: Blackmore
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D000071446:Neuronal Outgrowth / Q000235:genetics; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D051059:PTEN Phosphohydrolase / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection; D014404:Tubulin / Q000235:genetics / Q000378:metabolism
chemical_list: D009419:Nerve Tissue Proteins; D034741:RNA, Small Interfering; D014404:Tubulin; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D051059:PTEN Phosphohydrolase
keywords: CRISPR; Cortical neuron; High content screening; PTEN