High-content imaging-based pooled CRISPR screens in mammalian cells

Abstract

CRISPR (clustered regularly interspaced short palindromic repeats)-based gene inactivation provides a powerful means for linking genes to particular cellular phenotypes. CRISPR-based screening typically uses large genomic pools of single guide RNAs (sgRNAs). However, this approach is limited to phenotypes that can be enriched by chemical selection or FACS sorting. Here, we developed a microscopy-based approach, which we name optical enrichment, to select cells displaying a particular CRISPR-induced phenotype by automated imaging-based computation, mark them by photoactivation of an expressed photoactivatable fluorescent protein, and then isolate the fluorescent cells using fluorescence-activated cell sorting (FACS). A plugin was developed for the open source software μManager to automate the phenotypic identification and photoactivation of cells, allowing ∼1.5 million individual cells to be screened in 8 h. We used this approach to screen 6,092 sgRNAs targeting 544 genes for their effects on nuclear size regulation and identified 14 bona fide hits. These results present a scalable approach to facilitate imaging-based pooled CRISPR screens.

An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput CRISPRi screens

“…This presents an opportunity for molecular reporter development to contribute to the field of functional genomics. Several recent studies have combined pooled cell culturing with automated high-content microscopy, using in-place sequencing (Feldman et al, 2019;Wang et al, 2019), arrayed imaging (Wheeler et al, 2020), or photoinducible reporters (Kanfer et al, 2021;Yan et al, 2021) to identify hits. Such approaches simplify cell culturing and collection, but identifying phenotypic hits still requires time-consuming microscopy and computational analysis.…”

Chromatin mobility and relocation in DNA repair

“…While technological advances have enabled visualization of chromatin mobility and nuclear F-actin polymerization, these are typically time consuming live-imaging experiments not easily amenable to screening. However, screens that assess the molecular outcomes associated with nuclear F-actin polymerization, such as the increase in nuclear size that accompanies replication stress-induced nuclear F-actin [20], may provide opportunistic readouts enabling pathway dissection [101]. Similarly, directed interactomics using proximity biotinylation [102] provides a means to identify NPC or APB associated factors under conditions of specific genomic insult to be assessed thereafter through established cell and molecular biology approaches.…”

An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput screens

“…Presently, the biology which can be accessed by pooled-format screens is most limited by our ability to couple a cellular function of interest to a simple, robust readout. Several recent studies have combined pooled cell culturing with automated high-content microscopy, using in-place sequencing (Feldman et al, 2019;Wang et al, 2019), arrayed imaging (Wheeler et al, 2020), or photoinducible reporters (Kanfer et al, 2021;Yan et al, 2021) to identify hits. Such approaches simplify cell culturing and collection, but identifying phenotypic hits still requires time-consuming microscopy and computational analysis.…”