RNAi/CRISPR Screens: from a Pool to a Valid Hit

Schuster, Anne; Erasimus, Hélène; Fritah, Sabrina; Nazarov, Petr V.; Dyck, Eric Van; Niclou, Simone P.; Golebiewska, Anna

doi:10.1016/j.tibtech.2018.08.002

Cited by 95 publications

(88 citation statements)

References 105 publications

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“…Screens were also done in a simple and cost-effective process compared to arrayed screens. This highlights that our platform has the same advantage as other pooled screening methods (26). In addition, by employing scRNA-seq technology, we identified the perturbation as well as the signature of SNVs at the transcriptome level.…”

Section: Discussionmentioning

confidence: 74%

Single-cell analysis of a mutant library generated using CRISPR-guided deaminase

Jun

Lim

Chun³

et al. 2019

Preprint

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CRISPR-based screening methods using single-cell RNA sequencing (scRNA-seq) technology enable comprehensive profiling of gene perturbations from knock-out mutations. However, evaluating substitution mutations using scRNA-seq is currently limited. We combined CRISPR RNAguided deaminase and scRNA-seq technology to develop a platform for introducing mutations in multiple genes and assessing the mutationassociated signatures. Using this platform, we generated a library consisting of 420 sgRNAs, performed sgRNA tracking analysis, and assessed the effect size of the response to vemurafenib in the melanoma cell line, which has been well-studied via knockout-based drop-out screens. However, a substitution mutation library screen has not been applied and transcriptional information for mechanisms of action was not assessed. Our platform permits discrimination of several candidate mutations that function differently from other mutations by integrating sgRNA candidates and gene expression readout. We anticipate that our platform will enable high-throughput analyses of the mechanisms related to a variety of biological events.

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Section: Discussionmentioning

confidence: 74%

Single-cell analysis of a mutant library generated using CRISPR-guided deaminase

Jun

Lim

Chun³

et al. 2019

Preprint

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“…Before the advent of CRISPRi, RNAi was mostly used for gene knockdowns. In comparison to RNAi, which presents higher sequence‐specific off‐targets and varying knockdown efficiencies, CRISPRi‐mediated knockdown is considered a more secure gene perturbation technique [55]. Genome‐wide phenotypic screens using sgRNA libraries can be employed to identify novel protein functions by knocking down genes across a population of cells.…”

Section: Gaining Physiological Insights From Crispri Screensmentioning

confidence: 99%

Impact of CRISPR interference on strain development in biotechnology

Schultenkämper

Brito

Wendisch

2020

Biotech and App Biochem

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Genetic perturbation systems are of great interest to redirect metabolic fluxes for value‐added production, as well as genetic screening for the development of new drugs, or to identify new targets for biotechnological applications. Here, we review CRISPR interference (CRISPRi), a method for gene expression using a catalytically inactive version of the CRISPR‐associated protein 9 (dCas9) of the widely applied CRISPR‐Cas9 genome editing system. In combination with the appropriate sgRNA, dCas9 binds to specific DNA sequences without causing double‐stranded DNA breakage but interfering with transcription initiation or elongation. Besides manifold uses to interrogate the physiology of a bacterial cell, CRISPRi is used in applications for metabolic engineering and strain development in industrial biotechnology. Albeit in its infancy, CRISPRi has already delivered the first success stories; however, we also analyze limitations of the CRISPRi system and give future perspectives.

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“…For this analysis, we use MAGeCK MLE (using the maximum-likelihood estimate statistical approach; Li et al, 2014). Note that numerous analysis tools exist for CRISPR screening data, each employing different statistical approaches, and no consensus yet exists regarding best practices (Schuster et al, 2019). To use MAGeCK MLE, first use standard spreadsheet software to reorganize sample read counts into a single tab-delineated text file.…”

Section: Gene-level Statisticsmentioning

confidence: 99%

Pooled CRISPR Screens in Drosophila Cells

Viswanatha

Brathwaite

et al. 2019

CP Molecular Biology

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High‐throughput screens in Drosophila melanogaster cell lines have led to discovery of conserved gene functions related to signal transduction, host‐pathogen interactions, ion transport, and more. CRISPR/Cas9 technology has opened the door to new types of large‐scale cell‐based screens. Whereas array‐format screens require liquid handling automation and assay miniaturization, pooled‐format screens, in which reagents are introduced at random and in bulk, can be done in a standard lab setting. We provide a detailed protocol for conducting and evaluating genome‐wide CRISPR single guide RNA (sgRNA) pooled screens in Drosophila S2R+ cultured cells. Specifically, we provide step‐by‐step instructions for library design and production, optimization of cytotoxin‐based selection assays, genome‐scale screening, and data analysis. This type of project takes ∼3 months to complete. Results can be used in follow‐up studies performed in vivo in Drosophila, mammalian cells, and/or other systems. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Pooled‐format screening with Cas9‐expressing Drosophila S2R+ cells in the presence of cytotoxin Support Protocol 1: Optimization of cytotoxin concentration for Drosophila cell screening Support Protocol 2: CRISPR sgRNA library design and production for Drosophila cell screening Support Protocol 3: Barcode deconvolution and analysis of screening data

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RNAi/CRISPR Screens: from a Pool to a Valid Hit

Cited by 95 publications

References 105 publications

Single-cell analysis of a mutant library generated using CRISPR-guided deaminase

Single-cell analysis of a mutant library generated using CRISPR-guided deaminase

Impact of CRISPR interference on strain development in biotechnology

Pooled CRISPR Screens in Drosophila Cells

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