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SliceIt: A genome-wide resource and visualization tool to design CRISPR/Cas9 screens for editing protein-RNA interaction sites in the human genome.

ABSTRACT: Several protein-RNA cross linking protocols have been established in recent years to delineate the molecular interaction of an RNA Binding Protein (RBP) and its target RNAs. However, functional dissection of the role of the RBP binding sites in modulating the post-transcriptional fate of the target RNA remains challenging. CRISPR/Cas9 genome editing system is being commonly employed to perturb both coding and noncoding regions in the genome. With the advancements in genome-scale CRISPR/Cas9 screens, it is now possible to not only perturb specific binding sites but also probe the global impact of protein-RNA interaction sites across cell types. Here, we present SliceIt (http://sliceit.soic.iupui.edu/), a database of in silico sgRNA (single guide RNA) library to facilitate conducting such high throughput screens. SliceIt comprises of ~4.8 million unique sgRNAs with an estimated range of 2-8 sgRNAs designed per RBP binding site, for eCLIP experiments of >100 RBPs in HepG2 and K562 cell lines from the ENCODE project. SliceIt provides a user friendly environment, developed using advanced search engine framework, Elasticsearch. It is available in both table and genome browser views facilitating the easy navigation of RBP binding sites, designed sgRNAs, exon expression levels across 53 human tissues along with prevalence of SNPs and GWAS hits on binding sites. Exon expression profiles enable examination of locus specific changes proximal to the binding sites. Users can also upload custom tracks of various file formats directly onto genome browser, to navigate additional genomic features in the genome and compare with other types of omics profiles. All the binding site-centric information is dynamically accessible via "search by gene", "search by coordinates" and "search by RBP" options and readily available to download. Validation of the sgRNA library in SliceIt was performed by selecting RBP binding sites in Lipt1 gene and designing sgRNAs. Effect of CRISPR/Cas9 perturbations on the selected binding sites in HepG2 cell line, was confirmed based on altered proximal exon expression levels using qPCR, further supporting the utility of the resource to design experiments for perturbing protein-RNA interaction networks. Thus, SliceIt provides a one-stop repertoire of guide RNA library to perturb RBP binding sites, along with several layers of functional information to design both low and high throughput CRISPR/Cas9 screens, for studying the phenotypes and diseases associated with RBP binding sites.

SUBMITTER: Vemuri S

PROVIDER: S-EPMC7056568 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Bacillus strains are important industrial bacteria that can produce various biochemical products. However, low transformation efficiencies and a lack of effective genome editing tools have hindered its widespread application. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 techniques have been utilized in many organisms as genome editing tools because of their high efficiency and easy manipulation. In this study, an efficient genome editing method was developed for Bacillus licheniformis using a CRISPR-Cas9 nickase integrated into the genome of B. licheniformis DW2 with overexpression driven by the P43 promoter. The yvmC gene was deleted using the CRISPR-Cas9n technique with homology arms of 1.0 kb as a representative example, and an efficiency of 100% was achieved. In addition, two genes were simultaneously disrupted with an efficiency of 11.6%, and the large DNA fragment bacABC (42.7 kb) was deleted with an efficiency of 79.0%. Furthermore, the heterologous reporter gene aprN, which codes for nattokinase in Bacillus subtilis, was inserted into the chromosome of B. licheniformis with an efficiency of 76.5%. The activity of nattokinase in the DWc9nΔ7/pP43SNT-SsacC strain reached 59.7 fibrinolytic units (FU)/ml, which was 25.7% higher than that of DWc9n/pP43SNT-SsacC Finally, the engineered strain DWc9nΔ7 (Δepr ΔwprA Δmpr ΔaprE Δvpr ΔbprA ΔbacABC), with multiple disrupted genes, was constructed using the CRISPR-Cas9n technique. Taken together, we have developed an efficient genome editing tool based on CRISPR-Cas9n in B. licheniformis This tool could be applied to strain improvement for future research.IMPORTANCE As important industrial bacteria, Bacillus strains have attracted significant attention due to their production of biological products. However, genetic manipulation of these bacteria is difficult. The CRISPR-Cas9 system has been applied to genome editing in some bacteria, and CRISPR-Cas9n was proven to be an efficient and precise tool in previous reports. The significance of our research is the development of an efficient, more precise, and systematic genome editing method for single-gene deletion, multiple-gene disruption, large DNA fragment deletion, and single-gene integration in Bacillus licheniformis via Cas9 nickase. We also applied this method to the genetic engineering of the host strain for protein expression.

Dataset Information

SliceIt: A genome-wide resource and visualization tool to design CRISPR/Cas9 screens for editing protein-RNA interaction sites in the human genome.

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