Application of CRISPR genetic screens to investigate neurological diseases

Figures

• 

  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