name: inverse layout: true class: center, middle, inverse <div class="my-header"><span> <a href="/training-material/topics/genome-annotation" title="Return to topic page" ><i class="fa fa-level-up" aria-hidden="true"></i></a> <a class="nav-link" href="https://github.com/galaxyproject/training-material/edit/main/topics/genome-annotation/tutorials/crispr-screen/slides.html"><i class="fa fa-pencil" aria-hidden="true"></i></a> </span></div> <div class="my-footer"><span> <img src="/training-material/assets/images/GTN-60px.png" alt="Galaxy Training Network" style="height: 40px;"/> </span></div> --- <img src="/training-material/assets/images/GTN.png" alt="Galaxy Training Network" class="cover-logo"/> # Introduction to CRISPR screen analysis <div markdown="0"> <div class="contributors-line"> Authors: <a href="/training-material/hall-of-fame/mblue9/" class="contributor-badge contributor-mblue9"><img src="https://avatars.githubusercontent.com/mblue9?s=27" alt="Avatar">Maria Doyle</a> <a href="/training-material/hall-of-fame/sylviamahara/" class="contributor-badge contributor-sylviamahara"><img src="https://avatars.githubusercontent.com/sylviamahara?s=27" alt="Avatar">Sylvia Mahara</a> </div> </div> <!-- modified date --> <div class="footnote" style="bottom: 8em;"><i class="far fa-calendar" aria-hidden="true"></i><span class="visually-hidden">last_modification</span> Updated: Mar 10, 2022</div> <!-- other slide formats (video and plain-text) --> <div class="footnote" style="bottom: 6em;"> <i class="far fa-play-circle" aria-hidden="true"></i><span class="visually-hidden">video-slides</span> <a href="/training-material/videos/watch.html?v=/genome-annotation/tutorials/crispr-screen/slides">Video slides</a> | <i class="fas fa-file-alt" aria-hidden="true"></i><span class="visually-hidden">text-document</span><a href="slides-plain.html"> Plain-text slides</a> </div> <!-- usage tips --> <div class="footnote" style="bottom: 2em;"> <strong>Tip: </strong>press <kbd>P</kbd> to view the presenter notes | <i class="fa fa-arrows" aria-hidden="true"></i><span class="visually-hidden">arrow-keys</span> Use arrow keys to move between slides </div> ??? 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Useful when presenting. --- ## Requirements Before diving into this slide deck, we recommend you to have a look at: - [Introduction to Galaxy Analyses](/training-material/topics/introduction) --- ### <i class="far fa-question-circle" aria-hidden="true"></i><span class="visually-hidden">question</span> Questions - What is CRISPR? - What is a CRISPR screen? - How is a guide RNA library created? - What is the difference between a negative and positive screen? - How to analyse CRISPR screen data? --- ### <i class="fas fa-bullseye" aria-hidden="true"></i><span class="visually-hidden">objectives</span> Objectives - Describe what CRISPR screen data is - Outline how CRISPR screen data is generated and analysed --- ### What is CRISPR? .center[Overview of CRISPR knockout method] .image-100[] .pull-right[Adapted from [Addgene](https://www.addgene.org/guides/crispr/)] ??? - CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. - It's a bacterial immune system that has been modified for genome editing. - It consists of 2 components - a guide RNA and a non-specific CRISPR-associated endonuclease called Cas9. - The guide RNA is a short synthetic RNA composed of a scaffold sequence necessary for Cas9-binding and ~20 nucleotide spacer sequence that binds to the genomic target. - Cas9 induces a double-stranded break within the target DNA. - This results in in-frame amino acid deletions, insertions or frameshift mutations leading to premature stop codons within the targeted gene. - With CRISPR knockout methods, ideally the end result is a loss-of-function mutation within the targeted gene. - There are also CRISPR inhibition and activation methods but in this tutorial we focus on knockout. --- ### What is a CRISPR screen? .center[CRISPR screens enable high-throughput functional interrogation of a genome] .image-100[] ??? - The ease of generating guide RNAs makes CRISPR one of the most scalable genome editing technologies. - It can be used for genome-wide screens, enabling systematic targeting of tens of thousands of genes, with one gene targeted per cell. - With these screens we can identify the functions of genes, such as those essential for cell survival, drug resistance or sensitivity. - Pooled or arrayed screens can be performed. In this tutorial we analyse data from a pooled screen. --- ### How are the cells for the screen set up? .pull-right[ .image-60[] ] .pull-left[ * Various sgRNA libraries available e.g Brunello, Gecko, Yusa and [others](https://www.addgene.org/crispr/libraries/) * In this tutorial we use Brunello (77,441 sgRNAs: ~4 per human gene + 1000 non-targeting controls) * Cas9 expressing cells are transduced with sgRNA library * Whole genome screen should be carried out with min x300 guide representation ] ??? - Various guide RNA libraries are available and can be purchased. - In this tutorial we use Brunello, which is a human genome-wide CRISPR knockout library in a lentiGuide vector. - Cells expressing the Cas9 enzyme are transduced with the guide RNAs at a low Multiplicity of Infection (MOI), aiming for a minimum starting representation of 300 for each guide, and puromycin is used to remove cells without guides. --- ### What is the difference between a negative and positive screen? .image-100[] .pull-right[Adapted from [Addgene](https://www.addgene.org/guides/pooled-libraries/)] ??? - CRISPR positive or negative screens can be performed. - With a positive screen, few cells survive the treatment and we are interested in identifying genes whose guide RNAs increase (are enriched), indicating knockout of those genes leads to resistance. - With a negative screen, most cells survive after the treatment. In that case, we are interested in identifying genes whose guide RNAs decrease (are depleted) compared to a control e.g. vehicle, indicating knockout of those genes increases sensitivity to the treatment. - In this tutorial we analyse data from a negative screen. - Cells from the timepoints of interest are collected, genomic DNA is extracted, and the guide RNA region is amplified by PCR, followed by sequencing. --- ### A workflow for CRISPR screen analysis  ??? - This is an overview of the workflow used in the tutorial. - We first prepare the sequencing reads, checking quality and trimming adapters. - We then use the MAGeCK toolkit to analyse, followed by visualising results using volcano plots and identifying pathways. --- ## Thank You! This material is the result of a collaborative work. Thanks to the [Galaxy Training Network](https://training.galaxyproject.org) and all the contributors! <div markdown="0"> <div class="contributors-line"> Authors: <a href="/training-material/hall-of-fame/mblue9/" class="contributor-badge contributor-mblue9"><img src="https://avatars.githubusercontent.com/mblue9?s=27" alt="Avatar">Maria Doyle</a> <a href="/training-material/hall-of-fame/sylviamahara/" class="contributor-badge contributor-sylviamahara"><img src="https://avatars.githubusercontent.com/sylviamahara?s=27" alt="Avatar">Sylvia Mahara</a> </div> </div> <div style="display: flex;flex-direction: row;align-items: center;justify-content: center;"> <img src="/training-material/assets/images/GTN.png" alt="Galaxy Training Network" style="height: 100px;"/> </div> <a rel="license" href="https://creativecommons.org/licenses/by/4.0/"> This material is licensed under the Creative Commons Attribution 4.0 International License</a>.