Application of CRISPR genetic screens to investigate neurological diseases

Abstract

The adoption of CRISPR-Cas9 technology for functional genetic screens has been a transformative advance. Due to its modular nature, this technology can be customized to address a myriad of questions. To date, pooled, genome-scale studies have uncovered genes responsible for survival, proliferation, drug resistance, viral susceptibility, and many other functions. The technology has even been applied to the functional interrogation of the non-coding genome. However, applications of this technology to neurological diseases remain scarce. This shortfall motivated the assembly of a review that will hopefully help researchers moving in this direction find their footing. The emphasis here will be on design considerations and concepts underlying this methodology. We will highlight groundbreaking studies in the CRISPR-Cas9 functional genetics field and discuss strengths and limitations of this technology for neurological disease applications. Finally, we will provide practical guidance on navigating the many choices that need to be made when implementing a CRISPR-Cas9 functional genetic screen for the study of neurological diseases.

Keywords: CRISIPR KO; CRISPR-Cas9; CRISPRa; CRISPRi; Functional genetics; Marker selection screens; Neurodegenerative diseases; Neurological diseases; Survival screens; sgRNA.

Fig. 1

Overview of CRISPR-Cas9 functional genetics applications. Due to the inherently modular nature of CRISPR-Cas9 genome editing, there are many ways to implement a functional genetics screen based on this technology. Common choices realized in published work are highlighted in this figure in darker grey shading

Fig. 2

Workflow of CRISPR-Cas9 functional genetic screens. a sgRNA libraries are ligated onto plasmid backbones are then transformed into electrocompetent bacterial cells. The amplified sgRNA library is purified from a bacterial lysate and transfected into virus-producing cells to generate a sgRNA library. b The sgRNA library is transduced into target cells, which are subsequently subjected to phenotype selection. Genomic DNA is then harvested, and embedded sgRNAs are amplified by PCR and identified by NGS. Hits are determined and ranked by their relative enrichment or depletion of the respective sgRNAs in the selected versus non-selected control cells. c The initial validation of screen hits typically relies on: I. small-scale repeat analysis targeting genes of interest with sgRNAs that had been used in the original screen, plus additional sgRNAs directed toward the same gene; II. genomic sequencing-based verification that the targeted gene was indeed sequence-altered; and III. verification that restoring the wild-type gene sequence rescues the selection phenotype

Fig. 3

Notable firsts in the history of pooled, genome-scale CRISPR screens. a One of the first two CRISPR-Cas 9 KO screens searched for genes conferring vemurafenib resistance in melanoma cells [3]. b Subsequent CRISPR inhibition and activation (CRISPRi and CRISPRa) studies made use of deactivated Cas9 (dCas9) fused to repressor or activator domains for gene transcription modulation [50]. c A milestone in vivo study explored the role of a subset of genes in the evolution of metastatic tumors in an immunocompromised mouse [98]. d Primary cells were used in a study that employed tumor necrosis factor (Tnf) levels as a response marker to lipopolysaccharide treatment [70]. e A genome-scale CRISPRi screen on long, noncoding RNAs (lncRNAs) revealed that essential noncoding elements may be more cell-type specific than coding elements [99]

Fig. 4

Key considerations in choosing a screening method. Each research question poses a new set of challenges that need to be considered when selecting an appropriate screening method. This flowchart is intended to provide some initial guidance for investigators embarking on a CRISPR-Cas9 functional genomics screen regarding the choice of model and the type of screens that may be employed to address the neurological disease research question at hand