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# Introduction to CRISPR screen analysis
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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[![CRISPR method](../../images/crispr-screen/what_is_crispr.png)] .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[![CRISPR screen](../../images/crispr-screen/what_is_a_crispr_screen.png)] ??? - 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[![sgRNA library](../../images/crispr-screen/how_sgRNA_library_created.png)] ] .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[![Neg and pos screens](../../images/crispr-screen/pos_neg_screen.png)] .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 ![CRISPR workflow](../../images/crispr-screen/crispr_workflow.png) ??? - 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!
This material is licensed under the Creative Commons Attribution 4.0 International License