Drug Discovery


Figure 3


Arrayed CRISPR screening leverages the power of


synthetic crRNA to examine the specific effects of


modulation of a gene in a


single well. This can be done with something as simple as


survival or allow for extremely complex multiparametric high- content analysis to elucidate critical cellular behaviours.


genetic screening with single cell RNA sequencing (scRNA-seq)25-28. Perturb-seq, CRISP-seq and CROP-seq provide methods


that enable


researchers to directly link individual CRISPR- imposed genetic perturbations to a highly complex gene expression profile at the single cell level. Comparing the transcriptomes of many cells in parallel enables the comprehensive assessment of subpopulations within a heterogeneous group of cells and can provide mechanistic information on which genetic perturbations influence the pheno- types measured. These techniques even further expand the potential impact of CRISPR-based screens to a level where one can be unbiased in the scope of the phenotype evaluated as a consequence of each perturbation, creating the opportunity to study highly-complex diseases and exploit the ben- efits of pooled CRISPR screening analysis.


56


Arrayed phenotypic CRISPR screening Functional genomic screening in arrayed format offers the possibility to more directly explore com- plex functional assays or powerful phenotypic readouts, such as high-content analysis, that are less compatible with pooled screening approaches. Arrayed loss- or gain-of-function screens using CRISPRko and CRISPRa reagents offer alternative means to perturbing gene function compared to siRNA or cDNA overexpression reagents. An important distinction of CRISPRko/a to RNAi, however, is that cells also require Cas9 (or dCas9) present in the cells, adding an extra element to the screening procedure. This can be achieved by gen- erating a stable cell line with Cas9 using a lentivirus prior to screening, or by transient intro- duction of either Cas9 plasmid, mRNA or protein to the cells (see Figure 3).


Drug Discovery World Fall 2018


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