Introduction to CRISPR screen analysis
Contributors
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?
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]
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.pull-right[Adapted from Addgene]
Speaker Notes
- 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]
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Speaker Notes
- 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?
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Various sgRNA libraries available e.g Brunello, Gecko, Yusa and others
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In this tutorial we use Brunello (77,441 sgRNAs: ~4 per human gene + 1000 non-targeting controls)
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Cas9 expressing cells are transduced with sgRNA library
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Whole genome screen should be carried out with min x300 guide representation
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Speaker Notes
- 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]
Speaker Notes
- 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.
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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
Speaker Notes
- 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.