Quality control, modeling, and visualization of CRISPR screens with MAGeCK-VISPR

Li, Wei; Köster, Johannes; Han, Xu; Chen, Chen Hao; Xiao, Tengfei; Liu, Jun S.; Brown, Myles; Liu, X. Shirley

doi:10.1186/s13059-015-0843-6

Cited by 399 publications

(407 citation statements)

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“…We defined the negatively selected essential genes as those with significantly depleted sgRNAs at day 14 and positively selected genes as those with significantly enriched sgRNAs at day 14 (Dataset S1). MAGeCK and MAGeCK-VISPR are statistical algorithms we previously developed for CRISPR screen analyses (21,22). Whereas MAGeCK was developed to call genes from a single CRISPR screen experiment, MAGeCK-VISPR was developed to compare the gRNA abundance across many conditions.…”

Section: Resultsmentioning

confidence: 99%

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Genome-wide CRISPR screen identifies HNRNPL as a prostate cancer dependency regulating RNA splicing

Teng

Chen

Xiao

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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284

244

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Alternative RNA splicing plays an important role in cancer. To determine which factors involved in RNA processing are essential in prostate cancer, we performed a genome-wide CRISPR/Cas9 knockout screen to identify the genes that are required for prostate cancer growth. Functional annotation defined a set of essential spliceosome and RNA binding protein (RBP) genes, including most notably heterogeneous nuclear ribonucleoprotein L (HNRNPL). We defined the HNRNPL-bound RNA landscape by RNA immunoprecipitation coupled with next-generation sequencing and linked these RBP-RNA interactions to changes in RNA processing. HNRNPL directly regulates the alternative splicing of a set of RNAs, including those encoding the androgen receptor, the key lineage-specific prostate cancer oncogene. HNRNPL also regulates circular RNA formation via back splicing. Importantly, both HNRNPL and its RNA targets are aberrantly expressed in human prostate tumors, supporting their clinical relevance. Collectively, our data reveal HNRNPL and its RNA clients as players in prostate cancer growth and potential therapeutic targets.CRISPR screen | HNRNPL | prostate cancer | RNA binding protein | alternative splicing P rostate cancer is among the most prevalent adult malignancies in developed countries. The principal treatment for prostate cancer once it is no longer amenable to surgery or radiation treatment is androgen deprivation therapy, which targets androgen or androgen receptor (AR) signaling. However, resistance to androgen deprivation therapy often develops and leads to a state termed "castration-resistant prostate cancer," which still lacks an effective cure (1-3). Therefore, significant efforts have been devoted to better understand the mechanism of oncogenesis and to develop additional effective therapeutics targeting pivotal oncogenes, cancer-related signal transduction pathways, and epigenetic regulators (4, 5).Alternative RNA splicing is a fundamental cellular process by which a single gene can give rise to multiple different transcripts and proteins. This process is tightly regulated by core spliceosomes and other splicing factors, such as the serine/ arginine-rich family of proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs) (6, 7). Multiple studies indicate that deregulation of alternative splicing is implicated in cancer progression and that the splicing machinery may be targeted therapeutically (8-10). In addition to RNA splicing, the physical interactions between RNAs and RNA binding proteins (RBPs) underlie multiple RNA processing steps, such as capping, polyadenylation, transport, localization, modification, and translation, thereby regulating many aspects of RNA fate (11).Which RBPs and their related RNA processing steps are functionally important, especially in prostate cancer, remains elusive.The recent implementation of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease Cas9 genome editing system has proved effective in high-throughput lossof-function screens (12)(13)(14)...

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Section: Resultsmentioning

confidence: 99%

“…PCR was performed on genomic DNA to construct the sequencing library. Each library was sequenced at 30∼40 million reads to achieve ∼300× average coverage over the CRISPR library and data were analyzed by MAGeCK and MAGeCK-VISPR (21,22).…”

Section: Methodsmentioning

confidence: 99%

Genome-wide CRISPR screen identifies HNRNPL as a prostate cancer dependency regulating RNA splicing

Teng

Chen

Xiao

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

284

244

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“…Recently published tools for CRISPR pooled screen analysis, including CRISPRcloud (CC1) 27 , MAGeCK 25 , CarRpools 29 , CRISPRAnalyzeR 26 , and PinAPL-Py 28 , provide different methods for estimating the abundance of sgRNAs in each sample from pooled libraries. In most cases, input data consist of raw FASTQ-format sequencing result files.…”

Section: Algorithm For Quantifying Sgrna Abundance Previous Methods Amentioning

confidence: 99%

“…MAGeCK (Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout) was the first tool which was developed to provide CRISPR/Cas9 pooled screening data using a negativebinomial model and a modified robust ranking aggregation (RRA) algorithm 19 , and this allows deconvolution of the analysis from the beginning. HitSelect 20 , ScreenBEAM (Screening Bayesian Evaluation and Analysis Method) 21 , BAGEL (Bayesian Analysis of Gene Essentiality) 22 , sgRSEA (singleguide RNA Set Enrichment Analysis) 23 , PBNPA (Permutation based non-parametric analysis of CRISPR/Cas9 screen data) 24 , and MAGeCK-VISPR 25 proposed to provide a more accurate deconvolution of the data with different statistical models. However, those analysis tools tend to be script-based because they were developed for bioinformaticians or scientists who are very computationally savvy.…”

Section: Introductionmentioning

confidence: 99%

CRISPRCloud2: A cloud-based platform for deconvolving CRISPR screen data

Jeong

Kim

Rousseaux

et al. 2018

Preprint

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The simplicity and cost-effectiveness of CRISPR technology have made high-throughput pooled screening approaches available to many. However, the large amount of sequencing data derived from these studies yields often unwieldy datasets requiring considerable bioinformatic resources to deconvolute data; a feature which is simply not accessible to many wet labs. To address these needs, we have developed a cloud-based webtool CRISPRCloud2 that provides a state-of-the-art accuracy in mapping short reads to CRISPR library, a powerful statistical test that aggregates information across multiple sgRNAs targeting the same gene, a user-friendly data visualization and query interface, as well as easy linking to other CRISPR tools and bioinformatics resources for target prioritization. CRISPRCloud2 is a one-stop shop for labs analyzing CRISPR screen data.

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“…Genome editing is a powerful engineering tool that allows deliberate modification of a selected DNA sequence of the genome using site-specific nuclease with unprecedented accuracy. The technique has been revolusionized with the development of the more efficient and cost efective CRISPR/Cas9 system (Li et al, 2015;Fears and Meulen, 2017). Synthetic biology can be used to synthesize biologically-based complex systems that display new functions, including those new to nature (Serrano, 2007).…”

Section: Laboratory Biosecurity Risk Assessmentmentioning

confidence: 99%