Dataset Information

MicroRNA-focused CRISPR-Cas9 library screen reveals fitness-associated miRNAs.

ABSTRACT: MicroRNAs (miRNAs) are post-transcriptional gene regulators that play important roles in the control of cell fitness, differentiation, and development. The CRISPR-Cas9 gene-editing system is composed of the Cas9 nuclease in complex with a single guide RNA (sgRNA) and directs DNA cleavage at a predetermined site. Several CRISPR-Cas9 libraries have been constructed for genome-scale knockout screens of protein function; however, few libraries have included miRNA genes. Here we constructed a miRNA-focused CRISPR-Cas9 library that targets 1594 (85%) annotated human miRNA stem-loops. The sgRNAs in our LX-miR library are designed to have high on-target and low off-target activity, and each miRNA is targeted by four to five sgRNAs. We used this sgRNA library to screen for miRNAs that affect cell fitness of HeLa or NCI-N87 cells by monitoring the change in frequency of each sgRNA over time. By considering the expression in the tested cells and the dysregulation of the miRNAs in cancer specimens, we identified five HeLa pro-fitness and cervical cancer up-regulated miRNAs (miR-31-5p, miR-92b-3p, miR-146b-5p, miR-151a-3p, and miR-194-5p). Similarly, we identified six NCI-N87 pro-fitness and gastric cancer up-regulated miRNAs (miR-95-3p, miR-181a-5p, miR-188-5p, miR-196b-5p, miR-584-5p, and miR-1304-3p), as well as three anti-fitness and down-regulated miRNAs (let-7a-3p, miR-100-5p, and miR-149-5p). Some of those miRNAs are known to be oncogenic or tumor-suppressive, but others are novel. Taken together, the LX-miR library is useful for genome-wide unbiased screening to identify miRNAs important for cellular fitness and likely to be useful for other functional screens.

SUBMITTER: Kurata JS

PROVIDER: S-EPMC6004052 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

MicroRNA-focused CRISPR-Cas9 library screen reveals fitness-associated miRNAs.

Kurata Jessica S JS Lin Ren-Jang RJ

RNA (New York, N.Y.) 20180502 7

MicroRNAs (miRNAs) are post-transcriptional gene regulators that play important roles in the control of cell fitness, differentiation, and development. The CRISPR-Cas9 gene-editing system is composed of the Cas9 nuclease in complex with a single guide RNA (sgRNA) and directs DNA cleavage at a predetermined site. Several CRISPR-Cas9 libraries have been constructed for genome-scale knockout screens of protein function; however, few libraries have included miRNA genes. Here we constructed a miRNA-f ...[more]

PMID: 29720387

Similar Datasets

Project description:BackgroundCRISPR screens provide large-scale assessment of cellular gene functions. Pooled libraries typically consist of several single guide RNAs (sgRNAs) per gene, for a large number of genes, which are transduced in such a way that every cell receives at most one sgRNA, resulting in the disruption of a single gene in that cell. This approach is often used to investigate effects on cellular fitness, by measuring sgRNA abundance at different time points. Comparing gene knockout effects between different cell populations is challenging due to variable cell-type specific parameters and between replicates variation. Failure to take those into account can lead to inflated or false discoveries.ResultsWe propose a new, flexible approach called ShrinkCRISPR that can take into account multiple sources of variation. Impact on cellular fitness between conditions is inferred by using a mixed-effects model, which allows to test for gene-knockout effects while taking into account sgRNA-specific variation. Estimates are obtained using an empirical Bayesian approach. ShrinkCRISPR can be applied to a variety of experimental designs, including multiple factors. In simulation studies, we compared ShrinkCRISPR results with those of drugZ and MAGeCK, common methods used to detect differential effect on cell fitness. ShrinkCRISPR yielded as many true discoveries as drugZ using a paired screen design, and outperformed both drugZ and MAGeCK for an independent screen design. Although conservative, ShrinkCRISPR was the only approach that kept false discoveries under control at the desired level, for both designs. Using data from several publicly available screens, we showed that ShrinkCRISPR can take data for several time points into account simultaneously, helping to detect early and late differential effects.ConclusionsShrinkCRISPR is a robust and flexible approach, able to incorporate different sources of variations and to test for differential effect on cell fitness at the gene level. These improve power to find effects on cell fitness, while keeping multiple testing under the correct control level and helping to improve reproducibility. ShrinkCrispr can be applied to different study designs and incorporate multiple time points, making it a complete and reliable tool to analyze CRISPR screen data.

Project description:BackgroundThe development of lethal cancer metastasis depends on the dynamic interactions between cancer cells and the tumor microenvironment, both of which are embedded in the extracellular matrix (ECM). The acquisition of resistance to detachment-induced apoptosis, also known as anoikis, is a critical step in the metastatic cascade. Thus, a more in-depth and systematic analysis is needed to identify the key drivers of anoikis resistance.MethodsGenome-wide CRISPR/Cas9 knockout screen was used to identify critical drivers of anoikis resistance using SKOV3 cell line and found protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) as a candidate. Quantitative real-time PCR (qRT-PCR) and immune-histochemistry (IHC) were used to measure differentially expressed PCMT1 in primary tissues and metastatic cancer tissues. PCMT1 knockdown/knockout and overexpression were performed to investigate the functional role of PCMT1 in vitro and in vivo. The expression and regulation of PCMT1 and integrin-FAK-Src pathway were evaluated using immunoprecipitation followed by mass spectrometry (IP-MS), western blot analysis and live cell imaging.ResultsWe found that PCMT1 enhanced cell migration, adhesion, and spheroid formation in vitro. Interestingly, PCMT1 was released from ovarian cancer cells, and interacted with the ECM protein LAMB3, which binds to integrin and activates FAK-Src signaling to promote cancer progression. Strikingly, treatment with an antibody against extracellular PCMT1 effectively reduced ovarian cancer cell invasion and adhesion. Our in vivo results indicated that overexpression of PCMT1 led to increased ascites formation and distant metastasis, whereas knockout of PCMT1 had the opposite effect. Importantly, PCMT1 was highly expressed in late-stage metastatic tumors compared to early-stage primary tumors.ConclusionsThrough systematically identifying the drivers of anoikis resistance, we uncovered the contribution of PCMT1 to focal adhesion (FA) dynamics as well as cancer metastasis. Our study suggested that PCMT1 has the potential to be a therapeutic target in metastatic ovarian cancer.

Dataset Information

MicroRNA-focused CRISPR-Cas9 library screen reveals fitness-associated miRNAs.

Publications

MicroRNA-focused CRISPR-Cas9 library screen reveals fitness-associated miRNAs.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets