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1. FlyBase: updates to the Drosophila melanogaster knowledge base

   https://doi.org/10.1093/nar/gkaa1026  

   Larkin, Aoife ; Marygold, Steven J ; Antonazzo, Giulia ; Attrill, Helen ; dos Santos, Gilberto ; Garapati, Phani V ; Goodman, Joshua L ; Gramates, L Sian ; Millburn, Gillian ; Strelets, Victor B ; et al ( November 2020 , Nucleic Acids Research)

   null (Ed.)

   Abstract FlyBase (flybase.org) is an essential online database for researchers using Drosophila melanogaster as a model organism, facilitating access to a diverse array of information that includes genetic, molecular, genomic and reagent resources. Here, we describe the introduction of several new features at FlyBase, including Pathway Reports, paralog information, disease models based on orthology, customizable tables within reports and overview displays (‘ribbons’) of expression and disease data. We also describe a variety of recent important updates, including incorporation of a developmental proteome, upgrades to the GAL4 search tab, additional Experimental Tool Reports, migration to JBrowse for genome browsing and improvements to batch queries/downloads and the Fast-Track Your Paper tool. 

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2. Base Editing in Human Cells to Produce Single‐Nucleotide‐Variant Clonal Cell Lines

   https://doi.org/10.1002/cpmb.129  

   Vasquez, Carlos A. ; Cowan, Quinn T. ; Komor, Alexis C. ( November 2020 , Current Protocols in Molecular Biology)

   Base‐editing technologies enable the introduction of point mutations at targeted genomic sites in mammalian cells, with higher efficiency and precision than traditional genome‐editing methods that use DNA double‐strand breaks, such as zinc finger nucleases (ZFNs), transcription‐activator‐like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR‐associated protein 9 (CRISPR‐Cas9) system. This allows the generation of single‐nucleotide‐variant isogenic cell lines (i.e., cell lines whose genomic sequences differ from each other only at a single, edited nucleotide) in a more time‐ and resource‐effective manner. These single‐nucleotide‐variant clonal cell lines represent a powerful tool with which to assess the functional role of genetic variants in a native cellular context. Base editing can therefore facilitate genotype‐to‐phenotype studies in a controlled laboratory setting, with applications in both basic research and clinical applications. Here, we provide optimized protocols (including experimental design, methods, and analyses) to design base‐editing constructs, transfect adherent cells, quantify base‐editing efficiencies in bulk, and generate single‐nucleotide‐variant clonal cell lines. © 2020 Wiley Periodicals LLC.

   Basic Protocol 1: Design and production of plasmids for base‐editing experiments

   Basic Protocol 2: Transfection of adherent cells and harvesting of genomic DNA

   Basic Protocol 3: Genotyping of harvested cells using Sanger sequencing

   Alternate Protocol 1: Next‐generation sequencing to quantify base editing

   Basic Protocol 4: Single‐cell isolation of base‐edited cells using FACS

   Alternate Protocol 2: Single‐cell isolation of base‐edited cells using dilution plating

   Basic Protocol 5: Clonal expansion to generate isogenic cell lines and genotyping of clones

    

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3. FlyBase: updates to the Drosophila genes and genomes database

   https://doi.org/10.1093/genetics/iyad211  

   Öztürk-Çolak, Arzu ; Marygold, Steven J. ; Antonazzo, Giulia ; Attrill, Helen ; Goutte-Gattat, Damien ; Jenkins, Victoria K. ; Matthews, Beverley B. ; Millburn, Gillian ; dos Santos, Gilberto ; Tabone, Christopher J. ; et al ( February 2024 , GENETICS)

   FlyBase (flybase.org) is a model organism database and knowledge base about Drosophila melanogaster, commonly known as the fruit fly. Researchers from around the world rely on the genetic, genomic, and functional information available in FlyBase, as well as its tools to view and interrogate these data. In this article, we describe the latest developments and updates to FlyBase. These include the introduction of single-cell RNA sequencing data, improved content and display of functional information, updated orthology pipelines, new chemical reports, and enhancements to our outreach resources.

    

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4. Using Galaxy to Perform Large‐Scale Interactive Data Analyses—An Update

   https://doi.org/10.1002/cpz1.31  

   Ostrovsky, Alexander ; Hillman‐Jackson, Jennifer ; Bouvier, Dave ; Clements, Dave ; Afgan, Enis ; Blankenberg, Daniel ; Schatz, Michael C. ; Nekrutenko, Anton ; Taylor, James ; Team, the Galaxy ; et al ( February 2021 , Current Protocols)

   Modern biology continues to become increasingly computational. Datasets are becoming progressively larger, more complex, and more abundant. The computational savviness necessary to analyze these data creates an ongoing obstacle for experimental biologists. Galaxy (galaxyproject.org) provides access to computational biology tools in a web‐based interface. It also provides access to major public biological data repositories, allowing private data to be combined with public datasets. Galaxy is hosted on high‐capacity servers worldwide and is accessible for free, with an option to be installed locally. This article demonstrates how to employ Galaxy to perform biologically relevant analyses on publicly available datasets. These protocols use both standard and custom tools, serving as a tutorial and jumping‐off point for more intensive and/or more specific analyses using Galaxy. © 2021 Wiley Periodicals LLC.

   This article was corrected on 19 July 2022. See the end of the full text for details.

   Basic Protocol 1: Finding human coding exons with highest SNP density

   Basic Protocol 2: Calling peaks for ChIP‐seq data

   Basic Protocol 3: Compare datasets using genomic coordinates

   Basic Protocol 4: Working with multiple alignments

   Basic Protocol 5: Single cell RNA‐seq

    

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5. Programmable Gene Knockdown in Diverse Bacteria Using Mobile‐CRISPRi

   https://doi.org/10.1002/cpmc.130  

   Banta, Amy B. ; Ward, Ryan D. ; Tran, Jennifer S. ; Bacon, Emily E. ; Peters, Jason M. ( December 2020 , Current Protocols in Microbiology)

   Facile bacterial genome sequencing has unlocked a veritable treasure trove of novel genes awaiting functional exploration. To make the most of this opportunity requires powerful genetic tools that can target all genes in diverse bacteria. CRISPR interference (CRISPRi) is a programmable gene‐knockdown tool that uses an RNA‐protein complex comprised of a single guide RNA (sgRNA) and a catalytically inactive Cas9 nuclease (dCas9) to sterically block transcription of target genes. We previously developed a suite of modular CRISPRi systems that transfer by conjugation and integrate into the genomes of diverse bacteria, which we call Mobile‐CRISPRi. Here, we provide detailed protocols for the modification and transfer of Mobile‐CRISPRi vectors for the purpose of knocking down target genes in bacteria of interest. We further discuss strategies for optimizing Mobile‐CRISPRi knockdown, transfer, and integration. We cover the following basic protocols: sgRNA design, cloning new sgRNA spacers into Mobile‐CRISPRi vectors, Tn7transfer of Mobile‐CRISPRi to Gram‐negative bacteria, and ICEBs1transfer of Mobile‐CRISPRi to Bacillales. © 2020 The Authors.

   Basic Protocol 1: sgRNA design

   Basic Protocol 2: Cloning of new sgRNA spacers into Mobile‐CRISPRi vectors

   Basic Protocol 3: Tn7transfer of Mobile‐CRISPRi to Gram‐negative bacteria

   Basic Protocol 4: ICEBs1transfer of Mobile‐CRISPRi to Bacillales

   Support Protocol 1: Quantification of CRISPRi repression using fluorescent reporters

   Support Protocol 2: Testing for gene essentiality using CRISPRi spot assays on plates

   Support Protocol 3: Transformation ofE. coliby electroporation

   Support Protocol 4: Transformation of CaCl₂‐competentE. coli

    

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