6,755 research outputs found
PinAPL-Py: A comprehensive web-application for the analysis of CRISPR/Cas9 screens.
Large-scale genetic screens using CRISPR/Cas9 technology have emerged as a major tool for functional genomics. With its increased popularity, experimental biologists frequently acquire large sequencing datasets for which they often do not have an easy analysis option. While a few bioinformatic tools have been developed for this purpose, their utility is still hindered either due to limited functionality or the requirement of bioinformatic expertise. To make sequencing data analysis of CRISPR/Cas9 screens more accessible to a wide range of scientists, we developed a Platform-independent Analysis of Pooled Screens using Python (PinAPL-Py), which is operated as an intuitive web-service. PinAPL-Py implements state-of-the-art tools and statistical models, assembled in a comprehensive workflow covering sequence quality control, automated sgRNA sequence extraction, alignment, sgRNA enrichment/depletion analysis and gene ranking. The workflow is set up to use a variety of popular sgRNA libraries as well as custom libraries that can be easily uploaded. Various analysis options are offered, suitable to analyze a large variety of CRISPR/Cas9 screening experiments. Analysis output includes ranked lists of sgRNAs and genes, and publication-ready plots. PinAPL-Py helps to advance genome-wide screening efforts by combining comprehensive functionality with user-friendly implementation. PinAPL-Py is freely accessible at http://pinapl-py.ucsd.edu with instructions and test datasets
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Mitigation of off-target toxicity in CRISPR-Cas9 screens for essential non-coding elements.
Pooled CRISPR-Cas9 screens are a powerful method for functionally characterizing regulatory elements in the non-coding genome, but off-target effects in these experiments have not been systematically evaluated. Here, we investigate Cas9, dCas9, and CRISPRi/a off-target activity in screens for essential regulatory elements. The sgRNAs with the largest effects in genome-scale screens for essential CTCF loop anchors in K562 cells were not single guide RNAs (sgRNAs) that disrupted gene expression near the on-target CTCF anchor. Rather, these sgRNAs had high off-target activity that, while only weakly correlated with absolute off-target site number, could be predicted by the recently developed GuideScan specificity score. Screens conducted in parallel with CRISPRi/a, which do not induce double-stranded DNA breaks, revealed that a distinct set of off-targets also cause strong confounding fitness effects with these epigenome-editing tools. Promisingly, filtering of CRISPRi libraries using GuideScan specificity scores removed these confounded sgRNAs and enabled identification of essential regulatory elements
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Live cell imaging of low- and non-repetitive chromosome loci using CRISPR-Cas9.
Imaging chromatin dynamics is crucial to understand genome organization and its role in transcriptional regulation. Recently, the RNA-guidable feature of CRISPR-Cas9 has been utilized for imaging of chromatin within live cells. However, these methods are mostly applicable to highly repetitive regions, whereas imaging regions with low or no repeats remains as a challenge. To address this challenge, we design single-guide RNAs (sgRNAs) integrated with up to 16 MS2 binding motifs to enable robust fluorescent signal amplification. These engineered sgRNAs enable multicolour labelling of low-repeat-containing regions using a single sgRNA and of non-repetitive regions with as few as four unique sgRNAs. We achieve tracking of native chromatin loci throughout the cell cycle and determine differential positioning of transcriptionally active and inactive regions in the nucleus. These results demonstrate the feasibility of our approach to monitor the position and dynamics of both repetitive and non-repetitive genomic regions in live cells
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A CRISPR-based screen for Hedgehog signaling provides insights into ciliary function and ciliopathies.
Primary cilia organize Hedgehog signaling and shape embryonic development, and their dysregulation is the unifying cause of ciliopathies. We conducted a functional genomic screen for Hedgehog signaling by engineering antibiotic-based selection of Hedgehog-responsive cells and applying genome-wide CRISPR-mediated gene disruption. The screen can robustly identify factors required for ciliary signaling with few false positives or false negatives. Characterization of hit genes uncovered novel components of several ciliary structures, including a protein complex that contains δ-tubulin and ε-tubulin and is required for centriole maintenance. The screen also provides an unbiased tool for classifying ciliopathies and showed that many congenital heart disorders are caused by loss of ciliary signaling. Collectively, our study enables a systematic analysis of ciliary function and of ciliopathies, and also defines a versatile platform for dissecting signaling pathways through CRISPR-based screening
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CRISPR-Cas9-mediated functional dissection of 3'-UTRs.
Many studies using reporter assays have demonstrated that 3' untranslated regions (3'-UTRs) regulate gene expression by controlling mRNA stability and translation. Due to intrinsic limitations of heterologous reporter assays, we sought to develop a gene editing approach to investigate the regulatory activity of 3'-UTRs in their native context. We initially used dual-CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 targeting to delete DNA regions corresponding to nine chemokine 3'-UTRs that destabilized mRNA in a reporter assay. Targeting six chemokine 3'-UTRs increased chemokine mRNA levels as expected. However, targeting CXCL1, CXCL6 and CXCL8 3'-UTRs unexpectedly led to substantial mRNA decreases. Metabolic labeling assays showed that targeting these three 3'-UTRs increased mRNA stability, as predicted by the reporter assay, while also markedly decreasing transcription, demonstrating an unexpected role for 3'-UTR sequences in transcriptional regulation. We further show that CRISPR-Cas9 targeting of specific 3'-UTR elements can be used for modulating gene expression and for highly parallel localization of active 3'-UTR elements in the native context. Our work demonstrates the duality and complexity of 3'-UTR sequences in regulation of gene expression and provides a useful approach for modulating gene expression and for functional annotation of 3'-UTRs in the native context
Reactivating Fetal Hemoglobin Expression in Human Adult Erythroblasts Through BCL11A Knockdown Using Targeted Endonucleases.
We examined the efficiency, specificity, and mutational signatures of zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 systems designed to target the gene encoding the transcriptional repressor BCL11A, in human K562 cells and human CD34+ progenitor cells. ZFNs and TALENs were delivered as in vitro transcribed mRNA through electroporation; CRISPR/Cas9 was codelivered by Cas9 mRNA with plasmid-encoded guideRNA (gRNA) (pU6.g1) or in vitro transcribed gRNA (gR.1). Analyses of efficacy revealed that for these specific reagents and the delivery methods used, the ZFNs gave rise to more allelic disruption in the targeted locus compared to the TALENs and CRISPR/Cas9, which was associated with increased levels of fetal hemoglobin in erythroid cells produced in vitro from nuclease-treated CD34+ cells. Genome-wide analysis to evaluate the specificity of the nucleases revealed high specificity of this specific ZFN to the target site, while specific TALENs and CRISPRs evaluated showed off-target cleavage activity. ZFN gene-edited CD34+ cells had the capacity to engraft in NOD-PrkdcSCID-IL2Rγnull mice, while retaining multi-lineage potential, in contrast to TALEN gene-edited CD34+ cells. CRISPR engraftment levels mirrored the increased relative plasmid-mediated toxicity of pU6.g1/Cas9 in hematopoietic stem/progenitor cells (HSPCs), highlighting the value for the further improvements of CRISPR/Cas9 delivery in primary human HSPCs
CRISPR/Cas9‐mediated genome editing: from basic research to translational medicine
The recent development of the CRISPR/Cas9 system as an efficient and accessible programmable genome-editing tool has revolutionized basic science research. CRISPR/Cas9 system-based technologies have armed researchers with new powerful tools to unveil the impact of genetics on disease development by enabling the creation of precise cellular and animal models of human diseases. The therapeutic potential of these technologies is tremendous, particularly in gene therapy, in which a patient-specific mutation is genetically corrected in order to treat human diseases that are untreatable with conventional therapies. However, the translation of CRISPR/Cas9 into the clinics will be challenging, since we still need to improve the efficiency, specificity and delivery of this technology. In this review, we focus on several in vitro, in vivo and ex vivo applications of the CRISPR/Cas9 system in human disease-focused research, explore the potential of this technology in translational medicine and discuss some of the major challenges for its future use in patients.Portuguese Foundation for Science and Technology:
UID/BIM/04773/2013
1334
Spanish Ministry of Science, Innovation and Universities
RTI2018-094629-B-I00
Portuguese Foundation for Science and Technology
SFRH/BPD/100434/2014
European Union (EU)
748585
LPCC-NRS/Terry Fox grantsinfo:eu-repo/semantics/publishedVersio
Combinatorial CRISPR-Cas9 screens for de novo mapping of genetic interactions.
We developed a systematic approach to map human genetic networks by combinatorial CRISPR-Cas9 perturbations coupled to robust analysis of growth kinetics. We targeted all pairs of 73 cancer genes with dual guide RNAs in three cell lines, comprising 141,912 tests of interaction. Numerous therapeutically relevant interactions were identified, and these patterns replicated with combinatorial drugs at 75% precision. From these results, we anticipate that cellular context will be critical to synthetic-lethal therapies
Effects on the transcriptome upon deletion of a distal element cannot be predicted by the size of the H3K27Ac peak in human cells.
Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) associated with increased risk for colorectal cancer (CRC). A molecular understanding of the functional consequences of this genetic variation is complicated because most GWAS SNPs are located in non-coding regions. We used epigenomic information to identify H3K27Ac peaks in HCT116 colon cancer cells that harbor SNPs associated with an increased risk for CRC. Employing CRISPR/Cas9 nucleases, we deleted a CRC risk-associated H3K27Ac peak from HCT116 cells and observed large-scale changes in gene expression, resulting in decreased expression of many nearby genes. As a comparison, we showed that deletion of a robust H3K27Ac peak not associated with CRC had minimal effects on the transcriptome. Interestingly, although there is no H3K27Ac peak in HEK293 cells in the E7 region, deletion of this region in HEK293 cells decreased expression of several of the same genes that were downregulated in HCT116 cells, including the MYC oncogene. Accordingly, deletion of E7 causes changes in cell culture assays in HCT116 and HEK293 cells. In summary, we show that effects on the transcriptome upon deletion of a distal regulatory element cannot be predicted by the size or presence of an H3K27Ac peak
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