Introduction to CRISPR screen analysis

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# Introduction to CRISPR screen analysis

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				<a href="/training-material/hall-of-fame/mblue9/" class="contributor-badge contributor-mblue9"><img src="/training-material/assets/images/orcid.png" alt="orcid logo" width="36" height="36"/><img src="https://avatars.githubusercontent.com/mblue9?s=36" alt="Maria Doyle avatar" width="36" class="avatar" />
    Maria Doyle</a>
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				<a href="/training-material/hall-of-fame/sylviamahara/" class="contributor-badge contributor-sylviamahara"><img src="/training-material/assets/images/avatar.png" alt="Sylvia Mahara avatar" width="36" class="avatar" />
    Sylvia Mahara</a></li>
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## Requirements

Before diving into this slide deck, we recommend you to have a look at:

- [Introduction to Galaxy Analyses](/training-material/topics/introduction)

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### <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?

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### <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

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### 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/)]

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- 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.

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### 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)]

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- 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.

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### 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

]

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- 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.

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### 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/)]

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- 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.
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### A workflow for CRISPR screen analysis

![CRISPR workflow](../../images/crispr-screen/crispr_workflow.png)

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- 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.

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## 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!

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		<td><abbr title="These people wrote the bulk of the tutorial, they may have done the analysis, built the workflow, and wrote the text themselves.">Author(s)</abbr></td>
		<td>
			<a href="/training-material/hall-of-fame/mblue9/" class="contributor-badge contributor-mblue9"><img src="/training-material/assets/images/orcid.png" alt="orcid logo" width="36" height="36"/><img src="https://avatars.githubusercontent.com/mblue9?s=36" alt="Maria Doyle avatar" width="36" class="avatar" />
    Maria Doyle</a><a href="/training-material/hall-of-fame/sylviamahara/" class="contributor-badge contributor-sylviamahara"><img src="/training-material/assets/images/avatar.png" alt="Sylvia Mahara avatar" width="36" class="avatar" />
    Sylvia Mahara</a>
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		<td><abbr title="These people reviewed this material for accuracy and correctness">Reviewers</abbr></td>
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			<a href="/training-material/hall-of-fame/hexylena/" class="contributor-badge contributor-badge-small contributor-hexylena"><img src="https://avatars.githubusercontent.com/hexylena?s=36" alt="Helena Rasche avatar" width="36" class="avatar" /></a></td>
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Tutorial Content is licensed under <a rel="license" href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution 4.0 International License</a>.<br/>