Next-generation forward genetic screens: uniting high-throughput perturbations with single-cell analysis

Forward genetic screens coupling CRISPR (clustered regularly interspaced short palindromic repeats) perturbations with single-cell sequencing have rapidly advanced in the past few years. Recent improvements include increased scale of perturbations – from screening hundreds to now millions of cells – and multimodal phenotypic readouts beyond transcriptomes, such as open chromatin and cell-surface proteins.Analysis of CRISPR perturbation screens with single-cell sequencing requires connecting cells with the delivered perturbations. For this purpose, initial screens used barcodes delivered in tandem with CRISPR guide RNAs (gRNAs) but were plagued by high rates of barcode swapping. It is now possible to directly capture the functional CRISPR gRNAs, thus improving the fidelity and utility of these screens.Multimodal single-cell CRISPR screens represent a major development in the toolbox of the molecular geneticist, and enable high-throughput studies of cell function in healthy and disease states. Programmable genome-engineering technologies, such as CRISPR (clustered regularly interspaced short palindromic repeats) nucleases and massively parallel CRISPR screens that capitalize on this programmability, have transformed biomedical science. These screens connect genes and noncoding genome elements to disease-relevant phenotypes, but until recently have been limited to individual phenotypes such as growth or fluorescent reporters of gene expression. By pairing massively parallel screens with high-dimensional profiling of single-cell types/states, we can now measure how individual genetic perturbations or combinations of perturbations impact the cellular transcriptome, proteome, and epigenome. We review technologies that pair CRISPR screens with single-cell multiomics and the unique opportunities afforded by extending pooled screens using deep multimodal phenotyping. Programmable genome-engineering technologies, such as CRISPR (clustered regularly interspaced short palindromic repeats) nucleases and massively parallel CRISPR screens that capitalize on this programmability, have transformed biomedical science. These screens connect genes and noncoding genome elements to disease-relevant phenotypes, but until recently have been limited to individual phenotypes such as growth or fluorescent reporters of gene expression. By pairing massively parallel screens with high-dimensional profiling of single-cell types/states, we can now measure how individual genetic perturbat