CRISPR screen analysis

Overview

Questions:
  • What are the steps to process CRISPR screen data?

  • How to identify essential genes across experimental conditions?

Objectives:
  • Apply appropriate analysis and quality control steps for CRISPR screen data

  • Identify differentially enriched genes across conditions

  • Generate volcano plot to visualise results

Requirements:
Time estimation: 2 hours
Supporting Materials:
Last modification: Mar 9, 2022
License: Tutorial Content is licensed under Creative Commons Attribution 4.0 International License The GTN Framework is licensed under MIT

Introduction

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system is a bacterial immune system that has been modified for genome engineering. This groundbreaking technology resulted in a Nobel Prize for Emmanuelle Charpentier and Jennifer Doudna in 2020 (Uyhazi and Bennett 2021). CRISPR consists of two components: a guide RNA (gRNA) and a non-specific CRISPR-associated endonuclease (Cas9). The gRNA is a short synthetic RNA composed of a scaffold sequence necessary for Cas9-binding (trRNA) and ~20 nucleotide spacer or targeting sequence which defines the genomic target to be modified (crRNA). Cas9 induces double-stranded breaks (DSB) within the target DNA. The resulting DSB is then repaired by either error-prone Non-Homologous End Joining (NHEJ) pathway or less efficient but high-fidelity Homology Directed Repair (HDR) pathway. The NHEJ pathway is the most active repair mechanism and it leads to small nucleotide insertions or deletions (indels) at the DSB site. This results in in-frame amino acid deletions, insertions or frameshift mutations leading to premature stop codons within the open reading frame (ORF) of the targeted gene. Ideally, the end result is a loss-of-function mutation within the targeted gene; however, the strength of the knockout phenotype for a given mutant cell is ultimately determined by the amount of residual gene function.

The ease of generating gRNAs makes CRISPR one of the most scalable genome editing technologies and it has been recently utilized for genome-wide screens. These screens enable systematic targeting of 1000s of genes, with one gene targeted per cell, to identify genes driving phenotypes, such as cell survival, drug resistance or sensitivity. It is feasible for any laboratory to perform a CRISPR screen (Cluse et al. 2018) and they are being increasingly used to obtain biological insight (Bock et al. 2022, Przybyla and Gilbert 2021). These days, pooled whole-genome knockout, inhibition and activation CRISPR libraries and CRISPR sub-library pools are commonly screened.

Illustration of CRISPR Screen Method.
Figure 1: CRISPR knockout and activation methods (from Joung et al. 2016)

Agenda

In this tutorial, we will cover:

  1. Preparing the reads
    1. Data upload
    2. Raw reads QC
    3. Trim adapters
  2. Counting
  3. Testing
    1. Two conditions
    2. (Optional) Multiple conditions

Preparing the reads

Data upload

Here we will demonstrate analysing CRISPR screen using data from Fujihara et al. 2020. There are 3 samples from the human esophageal cancer cell line (OACM5.1): a baseline sample taken at time zero (T0-Control), a sample treated with drug for 8 days (T8-APR-246) and a control sample treated with vehicle for 8 days (T8-Vehicle). The aim is to identify genes whose knockout increases the cancer cells sensitivity to the drug. We will use FASTQ files containing 1% of reads from the original samples to demonstrate the read processing steps.

hands_on Hands-on: Retrieve CRISPR screen fastq datasets

  1. Create a new history for this tutorial
  2. Import the files from Zenodo:

    • Open the file galaxy-upload upload menu
    • Click on Rule-based tab
    • “Upload data as”: Collection(s)
    • Copy the following tabular data, paste it into the textbox and press Build

      T0-Control https://zenodo.org/api/files/6599878c-f569-41bf-a37a-2c6f3d2e67f9/T0-Control.fastq.gz
      T8-APR-246 https://zenodo.org/api/files/6599878c-f569-41bf-a37a-2c6f3d2e67f9/T8-APR-246.fastq.gz
      T8-Vehicle https://zenodo.org/api/files/6599878c-f569-41bf-a37a-2c6f3d2e67f9/T8-Vehicle.fastq.gz
      

    Rule-based Uploader.

    • From Rules menu select Add / Modify Column Definitions
      • Click Add Definition button and select List Identifier(s): column A

        tip Can’t find List Identifier?

        Then you’ve chosen to upload as a ‘dataset’ and not a ‘collection’. Close the upload menu, and restart the process, making sure you check Upload data as: Collection(s)

      • Click Add Definition button and select URL: column B

    • Click Apply
    • In the Name: box type fastqs and press Upload

    Rule-based Editor.

Raw reads QC

First we’ll check the quality of the raw read sequences with FastQC and aggregate the reports from the multiple samples with MultiQC (Ewels et al. 2016). We will check if the base quality is good and for presence of adapters. For more details on quality control and what the other FastQC plots mean see the “Quality control” tutorial. With CRISPR screens we expect adapter sequence to be present, surrounding the guide. The adapters for the Brunello library vector used with this dataset (lentiGuide-Puro) are shown below. We’ll get FASTQC to check for their presence by providing an adapters file with their sequences.

Adapter sequences dataset.
Figure 2: Reads from the T8-APR-246 sample with one of the guide sequences highlighted in blue (controlguide1 from Brunello library file). The adapter sequences are in red, directly adjacent to the guide on the right and left. The bases in purple at the start of the reads are stagger regions of different length (0-8bp), required to maintain diversity across the flowcell during sequencing of CRISPR screens.

details Adapters file

The adapters file (adapter_list.tsv) we use here with FASTQC was created by adding the two CRISPR adapter sequences (5’ and 3’ of the guide) to the bottom of the adapters file used by FASTQC here. The first 12 bases of the CRISPR adapters were used, as that length is currently recommended in the FASTQC adapters file.

hands_on Hands-on: Quality control

  1. Import the adapters file from Zenodo or the Shared Data library (if available):
    https://zenodo.org/api/files/6599878c-f569-41bf-a37a-2c6f3d2e67f9/adapter_list.tsv
    
    • Copy the link location
    • Open the Galaxy Upload Manager (galaxy-upload on the top-right of the tool panel)

    • Select Paste/Fetch Data
    • Paste the link into the text field

    • Press Start

    • Close the window
  2. FastQC Tool: toolshed.g2.bx.psu.edu/repos/devteam/fastqc/fastqc/0.73+galaxy0 with the following parameters:
    • param-collection “Short read data from your current history”: fastqs (click “Dataset collection” button on left-side of this input field)
    • param-file“Adapter list”: adapter_list.tsv
  3. MultiQC Tool: toolshed.g2.bx.psu.edu/repos/iuc/multiqc/multiqc/1.11+galaxy0 with the following parameters to aggregate the FastQC reports:
    • In “Results”
      • “Which tool was used generate logs?”: FastQC
      • In “FastQC output”
        • “Type of FastQC output?”: Raw data
        • param-collection “FastQC output”: Raw data files (output of FastQC)
  4. Add a tag (#fastqc-untrimmed) to the MultiQC webpage to make it easy to differentiate this dataset in the History from other MultiQC reports we will generate.

    Tip: Adding a tag

    • Click on the dataset
    • Click on galaxy-tags Edit dataset tags
    • Add a tag starting with #

      Tags starting with # will be automatically propagated to the outputs of tools using this dataset.

    • Check that the tag is appearing below the dataset name
  5. Inspect the webpage output from MultiQC

    question Questions

    1. What is the read length?
    2. What do you think of the base quality of the sequences?
    3. Are adapters detected?

    solution Solution

    1. The read length is 75 bp.
    2. The base quality is good for the 3 files, as can be seen in the plot below.

      Sequence quality.

    3. Yes. In the Adapter Content plot (below) we can see the two CRISPR adapters have been detected. The adapter 5 prime of the sgRNA starts appearing at the beginning of the reads and has been detected in nearly 100% of reads. The adapter 3 prime of the sgRNA appears after base 40. There’s also a little Illumina Universal adapter detected towards the ends of some reads which indicate the presence of short fragments, possibly adapter dimers.

      Adapter content.

Trim adapters

We’ll trim the adapters from these sequences using Cutadapt (Marcel 2011). To trim, we’ll use the 5’ adapter sequence. We don’t need to trim the 3’ adapter as Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MAGeCK) will only use the first 20bp from each read. It determines the number of bases to use automatically from the length of the sequences in the library file, in our case 20bp, or we can specify the sgRNA length.

details Adapter trimming

In this dataset the adapters start at different positions in the reads, as was shown above. MAGeCK count can trim adapters around the guide sequences. However, the adapters need to start at the same position in each read, requiring the same trimming length, as described on the MAGeCK website here. An example for what MAGeCK expects is shown below. If you used MAGeCK count trimming with the dataset in this tutorial it wouldn’t be able to trim the 5’ adapter properly and you would only get ~60% reads mapping instead of >80%.

Adapters MAGeCK can trim.
Figure 3: Example showing what MAGeCK count expects to be able to auto-detect and trim adapters. Guide sequence is higlighted in blue with the adapter sequences directly adjacent on the right and left. MAGeCK count uses the first 20 bases of each read to map so the sequence after the guide is less important to trim exactly.

For this dataset, as the adapters are not at the same position in each read, we need to trim the adapters before running MAGeCK count.

hands_on Hands-on: Trim adapters

  1. Cutadapt Tool: toolshed.g2.bx.psu.edu/repos/lparsons/cutadapt/cutadapt/3.5+galaxy0 with the following parameters:
    • “Single-end or Paired-end reads?”: Single-end
      • param-collection “FASTQ/A file #1”: all fastq.gz files
      • In “Read 1 Options”:
        • In “5’ (End) Adapters”:
          • param-repeat “Insert 5’ (Front) Adapters”
            • “Source”: Enter custom sequence
              • “Enter custom 5’ adapter sequence”: TTGTGGAAAGGACGAAACACCG
    • “Outputs selector”:
      • “Report”: tick
  2. Inspect the Cutadapt report

    question Questions

    For sample T8-APR-246:

    What % of reads contained adapter?

    solution Solution

    99.6%

  3. MultiQC Tool: toolshed.g2.bx.psu.edu/repos/iuc/multiqc/multiqc/1.11+galaxy0 with the following parameters to aggregate the Cutadapt reports:
    • In “Results”
      • “Which tool was used generate logs?”: Cutadapt/Trim Galore!
        • param-collection “Output of Cutadapt”: Report files (output of Cutadapt)
  4. Add a tag (#cutadapt-report) to the MultiQC webpage to differentiate this report from the previous (FastQC) one

  5. Inspect the MultiQC report

MultiQC produces a 5’ trimmed sequences plot where we can check the results are as expected. Here we can see the length of sequence trimmed from the start of the read ranges from 22bp to 30bp with a dip at 27bp. This corresponds to the length of the adapter (22bp) plus stagger sequence (0,1,2,3,4,6,7,8bp) (see sequencing protocol). There is no 5bp stagger sequence so 27bp sequences are not expected to be trimmed. The trimmed sequence lengths are what we expect for this dataset and the plot looks similar for all our samples which is good.

Plot showing lengths of sequences trimmed from 5' end of reads.

hands_on Exercise: Quality control of the polished datasets

Use FastQC Tool: toolshed.g2.bx.psu.edu/repos/devteam/fastqc/fastqc/0.73+galaxy0 and MultiQC Tool: toolshed.g2.bx.psu.edu/repos/iuc/multiqc/multiqc/1.11+galaxy0 like before, but using the the trimmed datasets produced by Cutadapt as input.

question Questions

How did read trimming affect the Adapter Content plot?

solution Solution

In the Adapter Content section we now don’t have any 5’ adapter detected. The first 20bp of the reads, what MAGeCK will use for our dataset, has no adapter detected.

Plot of trimmed sequences showing no adapter detected in first 20 bases.

Counting

For the rest of the CRISPR screen analysis, counting and testing, we’ll use the tool called MAGeCK (Li et al. 2014, Li et al. 2015).

To count how many guides we have for each gene, we need a library file that tells us which guide sequence belongs to which gene. The guides used here are from the Brunello library (Doench et al. 2016) which contains 77,441 sgRNAs, an average of 4 sgRNAs per gene, and 1000 non-targeting control sgRNAs. The library file must be tab-separated and contain no spaces within the gene or target names. If necessary, there are tools in Galaxy that can format the file removing spaces and converting commas to tabs.

hands_on Hands-on: Count guides per gene

  1. Import the sgRNA library file
    https://zenodo.org/api/files/6599878c-f569-41bf-a37a-2c6f3d2e67f9/brunello.tsv
    
  2. MAGeCK count Tool: toolshed.g2.bx.psu.edu/repos/iuc/mageck_count/mageck_count/0.5.9.2.4 with the following parameters:
    • Reads Files or Count Table?”: Separate Reads files
      • param-collection “Sample reads: the Read 1 Output (outputs of Cutadapt)
    • param-file “sgRNA library file”: the brunello.tsv file
    • In “Output Options”:
      • “Output Count Summary file”: Yes
      • “Output plots”: Yes
  3. We have been using 1% of reads from the samples. Import the MAGeCK count files (sgRNA counts, counts summary and plots pdf) for the full dataset so you can see what results for a real dataset looks like.
    https://zenodo.org/api/files/6599878c-f569-41bf-a37a-2c6f3d2e67f9/kenji_mageck_sgrna_counts.tsv
    https://zenodo.org/api/files/6599878c-f569-41bf-a37a-2c6f3d2e67f9/kenji_mageck_count_summary.tsv
    https://zenodo.org/api/files/6599878c-f569-41bf-a37a-2c6f3d2e67f9/kenji_mageck_count_report.pdf
    

MAGeCK count outputs:

  • a sgRNA Counts file
  • a Count Summary file
  • a PDF report

Count Summary file

The contents of the count summary file is explained on the MAGeCK website here, also shown below. The columns are as follows. To help you evaluate the quality of the data, recommended values from the MAGeCK authors are shown in bold.

Column Content
File The fastq (or the count table) file used.
Label The label of that fastq file assigned.
Reads Total number reads in the fastq file. (Recommended: 100~300 times the number of sgRNAs)
Mapped Total number of reads that can be mapped to library
Percentage Mapped percentage, calculated as Mapped/Reads (Recommended: at least 60%)
TotalsgRNAs Total number of sgRNAs in the library
Zerocounts Total number of missing sgRNAs (sgRNAs that have 0 counts) (Recommended: no more than 1%)
GiniIndex The Gini Index of the read count distribution. A smaller value indicates more eveness of the count distribution. (Recommended: around 0.1 for plasmid or initial state samples, and around 0.2-0.3 for negative selection samples)

details Gini index

Gini index is a measure of inequality from economics. It is used in CRISPR analysis to assess if sgRNAs are present in equal amounts. In positive selection experiments, where only some sgRNAs dominate, the index can be high. However, as discussed in Li et al. 2015, in plasmid library, in early time points, or negative selection experiments, we expect fairly even distribution of the remaining sgRNAs that haven’t been negatively selected. A high Gini index in these types of sample can indicate CRISPR oligonucleotide synthesis unevenness, low viral transfection efficiency, and overselection, respectively.

question Questions

Is the data quality good for the 3 samples? Use the count summary file for the full dataset, and the recommended values in the table above, to answer these questions.

  1. Have we sequenced enough reads?
  2. Is the mapped percentage good?
  3. Is the sgRNA zero count value good?
  4. Is the Gini Index good?

solution Solution

  1. The number of reads is ok. For example, for T0 control sample we have 17,272,052 reads mapped to guides. We have 77,441 guides so we have ~220 reads per guide (17,272,052/77,441). A minimum of 100 reads per guide, preferably 300, is recommended.
  2. Yes, in the summary we have >85% mapped for all 3 samples. MAGeCK count does not allow any base mismatches between the reads and the library file, as described here so we expect not all reads will map. Note that we filtered out (6-10%) reads with Cutadapt so we should include those in our unmapped % if we want an accurate count.
  3. T0-Control has 0.71% (546/77441 * 100) sgRNAs that have no reads mapped, which is good. The T8 samples are just slightly high at 2.3% (1752/77441 * 100) and 2.8% (2170/77441 * 100).
  4. The Gini Index is 0.09 for T0-Control (initial state) which is good. The T8 samples are higher at 0.13 and 0.14 but good for a negative selection experiment.

MAGeCK count can also generate a PDF with plots that can help assess quality.

hands_on Hands-on: Assess mageck count plots

Inspect the PDF we imported above.

question Questions

What can you determine about the samples from the plots?

solution Solution

In the boxplots, we can see we have largely similiar distributions of counts for the 3 samples. The greater length of the box and between whiskers in the T8 samples compared to the control tells us we have a bit more variability of counts in those samples, more guides with low and high counts in T8 compared to the T0. MAGeCK count boxplots.

The Distribution of read counts plot shows us how many guides we have for each count (the frequency sums to the total no. of guides 77,441). Similar to the boxplots, the wider distribution for T8 compared to T0 shows us that those samples have more guides with low and high counts. The peak for T0 appears lower just because it has more points (bins) in the plot. MAGeCK count histograms.

The PCA plot shows us the samples from the different conditions separate well. The T8 samples are a bit more similar to each other, to each other on PC1 axis than to T0. If we had more samples we could use this plot to check for clustering of replicates, batch effect or outliers. MAGeCK count PCA plot.

The hierarchical clustering plot also shows us that the T8 samples are a bit more similar to each other than to T0. MAGeCK count hierarchical clustering plot.

The paper by Li et al. 2015 has more information on MAGeCK quality control.

Testing

Now that we’ve generated our guide counts, we’ll use MAGeCK test to identify essential genes. Essential means positively or negatively selected sgRNAs and genes. CRISPR positive or negative selection screens can be performed. With a positive selection screen, most cells die after the treatment (selection) and we are interested in identifying genes whose sgRNAs increase and dominate, indicating loss of those genes helps cells survive that treatment. This can help identify genes essential for drug resistance. With a negative selection screen, most cells survive after the treatment. In that case, we are interested in identifying genes whose sgRNAs decrease (drop out) compared to a control (e.g. vehicle), indicating those genes are needed for the cells to survive with that treatment. This can help identify genes essential for drug sensitivity. Regardless of the type of screen performed (positive or negative), MAGeCK can identify both positively and negatively selected genes in the screen (Li et al. 2014). The dataset we are using in this tutorial is from a negative selection screen where the aim is to identify genes whose knockout increases the cancer cells sensitivity to the drug.

Positive and negative selection screens.
Figure 4: Adapted from Addgene

Two conditions

If we want to compare the drug treatment (T8-APR-246) to the vehicle control (T8-Vehicle) we can use MAGeCK test. MAGeCK test uses a robust ranking aggregation (RRA) algorithm (Li et al. 2014).

Diagram of MAGeCK RRA algorithm.
Figure 5: Overview of the MAGeCK algorithm. Raw read counts corresponding to single-guided RNAs (sgRNAs) from different experiments are first normalized using median normalization and mean-variance modeling is used to capture the relationship of mean and variance in replicates. The statistical significance of each sgRNA is calculated using the learned mean-variance model. Essential genes (both positively and negatively selected) are then identified by looking for genes whose sgRNAs are ranked consistently higher (by significance) using robust rank aggregation (RRA) (from Li et al. 2014)

hands_on Hands-on: Test for enrichment

  1. MAGeCKs test Tool: toolshed.g2.bx.psu.edu/repos/iuc/mageck_test/mageck_test/0.5.9.2.1 with the following parameters:
    • param-file “Counts file”: kenji_mageck_sgRNA_counts.tsv(the counts file for the full dataset that we imported)
    • “Specify Treated samples or Control”: Treated samples
      • “Treated Sample Labels (or Indexes)”: 0
    • “Control Sample Labels (or Indexes)”: 1
    • In “Output Options”:
      • “Output normalized counts file”: Yes
      • “Output plots”: Yes

details MAGeCK test sample names

For MAGeCK test we could specify the samples using their names, which must match the names used in the columns of the counts file, but the hyphens we have in our sample names aren’t allowed. We could change the hyphens in the counts file or we can specify the samples by their positions in the counts file, with the first sample column being 0. We do that here.

comment Replicates

If we have biological and/or technical replicates we can handle them in a similar way to that described on the MAGeCK website. For biological replicates, we input them in MAGeCK test Treated Sample Labels/Control Sample Labels fields separated by a comma. For technical replicates, we could combine the fastqs for each sample/biological replicate, for example with the Concatenate datasets tool, before running MAGeCK count.

details Normalization

We are using MAGeCK’s default normalization method “median” which is more robust to outliers. Figure M1 from Li et al. 2014 shows a comparison of median (“median”) versus total (“total”) normalization for two CRISPR screen datasets. The distribution of the read counts of significant sgRNAs (FDR=1%) was compared with the mean read count distribution of all sgRNAs (“all”, black). The distribution of the significant sgRNAs should be similar to the distribution of all sgRNAs if the normalization method is unbiased. The difference is small for the leukemia dataset. However, in the melanoma dataset, where a few sgRNAs have very large read counts, the difference is larger, as “total” normalization will prefer sgRNAs with higher read-counts. In contrast, the distribution after “median” normalization is closer to the distribution of all sgRNAs.

Median versus Total normalization.

If many genes have zero counts, such as after treatment in a positive selection screen, the median may be zero and we may get a message in the MAGeCK log that we have to use total normalization.

Instead of median or total, we could choose to normalize using control guide sgRNAs. However, we would need to know that they’re not changing due to the experiment or introducing bias (Chen et al. 2018).

MAGeCK test outputs:

  • a Gene Summary file
  • a sgRNA Summary file
  • a PDF report

Gene Summary file

The Gene Summary file contains the columns described below and a row for each gene targeted by sgRNAs. We have >20,000 genes in the file for this dataset. We get values for both negative and positive selection. The dataset here is from a negative selection screen so we are most interested in the negative values. Genes are ranked by the p.neg field (by default). If you need a ranking by the p.pos, you can use the Sort data in ascending or descending order tool in Galaxy.

Column name Content
id Gene ID
num The number of targeting sgRNAs for each gene
neg|score The RRA lo value of this gene in negative selection
neg|p-value The raw p-value (using permutation) of this gene in negative selection
neg|fdr The false discovery rate of this gene in negative selection
neg|rank The ranking of this gene in negative selection
neg|goodsgrna The number of “good” sgRNAs, i.e., sgRNAs whose ranking is below the alpha cutoff (determined by the –gene-test-fdr-threshold option), in negative selection.
neg|lfc The log2 fold change of this gene in negative selection. The way to calculate gene lfc is controlled by the –gene-lfc-method option
pos|score The RRA lo value of this gene in positive selection
pos|p-value The raw p-value (using permutation) of this gene in positive selection
pos|fdr The false discovery rate of this gene in positive selection
pos|rank The ranking of this gene in positive selection
pos|goodsgrna The number of “good” sgRNAs, i.e., sgRNAs whose ranking is below the alpha cutoff (determined by the –gene-test-fdr-threshold option), in positive selection.
pos|lfc The log fold change of this gene in positive selection

sgRNA summary file

The sgRNA Summary file contains the columns described below. We can use the sgRNA file to check how the individual guides for genes of interest performed.

Column Content
sgrna sgRNA ID
Gene The targeting gene
control_count Normalized read counts in control samples
treatment_count Normalized read counts in treatment samples
control_mean Median read counts in control samples
treat_mean Median read counts in treatment samples
LFC The log2 fold change of sgRNA
control_var The raw variance in control samples
adj_var The adjusted variance in control samples
score The score of this sgRNA
p.low p-value (lower tail)
p.high p-value (higher tail)
p.twosided p-value (two sided)
FDR false discovery rate
high_in_treatment Whether the abundance is higher in treatment samples

PDF report

The PDF shows plots of the top 10 negatively and positively selected genes. We can see the top genes ranked by RRA scores or p value. These values come from the gene summary file.

MAGeCK test RRA scores.

question Questions

What are the top 3 negatively selected genes by P value?

solution Solution

ESD, MTHFD1L and SHMT2, the same as by RRA score. RRA score is like p-value. Those genes are part of the glutathione pathway which was found to be altered in the published paper for this dataset.

MAGeCK test pvalues.

The PDF also shows plots with the sgRNA counts for the top 10 genes. These values are the normalized counts for each sgRNA from the sgRNA summary file. With these plots we can see if the counts of all the sgRNAs for these top genes are changing similarly.

Plot showing guide counts for ESD gene.

question Questions

  1. Are all the sgRNAs for FLI1 (the 4th top neg gene) changing similarly?
  2. In the gene summary table, how many of the negatively selected genes have FDR < 0.05?

solution Solution

  1. No. We can see in this case that, while one sgRNA is a lot lower in the APR treated sample compared to the vehicle, one increases a little, and the other two sgRNAs don’t change much. So we might conclude that this gene is not strongly negatively selected.
  2. None. One reason for this is likely the large number of genes being tested (>20,000). You could try to increase sensitivity with the procedures described here.

Plot showing guide counts for FLI1 gene.

Visualisation

In addition to the visualisations automatically generated by MAGeCK in the PDF, we can create a volcano plot to further visualise the output. With a volcano plot we plot the magnitude of change for drug treatment versus vehicle control (lfc) versus significance (p-value). As we have two columns for lfc and p-value, one for negative selection and one for positive, we first combine these into one column for each using the awk tool. If the neg|p-value is smaller than the pos|p-value the gene is negatively selected. If the neg|p-value is larger than the pos|p-value the gene is positively selected. Then we create the plot using the Volcano plot tool.

hands_on Hands-on: Create volcano plot

  1. Text reformatting with awk Tool: toolshed.g2.bx.psu.edu/repos/bgruening/text_processing/tp_awk_tool/1.1.2 with the following parameters:
    • param-file “File to process”: MAGeCK test Gene Summary
    • “AWK Program”: Copy and paste the text in the grey box below into this field
    # Print new header for first line
    NR == 1 { print "gene", "pval", "fdr", "lfc" }
    
    # Only process lines after first
    NR > 1 {
        # check if neg pval (column 4) is less than pos pval (column 10)
        if ($4 < $10){
           # if it is, print negative selection values
            print $1, $4, $5, $8
        } else {
           # if it's not, print positive selection values
            print $1, $10, $11, $14
        }
    }
    
  2. Inspect the file output. It should look like below.
    gene    pval        fdr       lfc
    ESD     1.9454e-05  0.324257  -1.9283
    MTHFD1L 3.226e-05   0.324257  -0.89778
    SHMT2   7.6093e-05  0.454208  -1.1328
    FLI1    9.0376e-05  0.454208  -0.08872
    
  3. Volcano Plot Tool: toolshed.g2.bx.psu.edu/repos/iuc/volcanoplot/volcanoplot/0.0.5 to create a volcano plot
    • param-file “Specify an input file”: the Text reformatting output file
    • param-file “File has header?”: Yes
    • param-select “FDR (adjusted P value)”: Column 3
    • param-select “P value (raw)”: Column 2
    • param-select “Log Fold Change”: Column 4
    • param-select “Labels”: Column 1
    • param-select “Points to label”: Significant
      • param-text “Only label top most significant”: 10
  4. Inspect the plot in the PDF output.

    question Questions

    What is the most significant gene?

    solution Solution

    ATP5E as it is the gene nearest the top of the plot.

    Volcano plot for APR compared to vehicle.

For more details on using the volcano plot tool, see the tutorial here. For how to customise the volcano plot tool output using R, see the tutorial here.

Pathway analysis

We can perform pathway analysis on the results to identify pathways that are changing with the treatment. MAGeCK has a pathway analysis module, however, we will use the tool fgsea as it outputs some visualisations and the genes enriched in the pathway. For fgsea we need a ranked list of genes and a pathways file in GMT format. We can get the ranked list from our mageck results, we’ll use the RRA score column for negative results. For the pathways, we’ll use the Hallmark pathways from MSigDB, which can be a good starting point for pathway exploration as discussed here.

hands_on Hands-on: Perform gene set enrichment with fgsea

  1. Import the Hallmark pathways file Zenodo or the Shared Data library (if available). Set the Type to tabular:
    https://zenodo.org/api/files/6599878c-f569-41bf-a37a-2c6f3d2e67f9/h.all.v7.4.symbols.gmt
    

    Tip: Changing the datatype

    • Click on the galaxy-pencil pencil icon for the dataset to edit its attributes
    • In the central panel, click on the galaxy-chart-select-data Datatypes tab on the top
    • Select your desired datatype
    • Click the Save button
  2. Cut columns from a table tool with
    • param-text “Cut columns”: c1,c3 (the gene symbols and neg score)
    • param-select “Delimited by”: Tab
    • param-file “From”: the MAGeCK Gene Summary file
  3. fgsea tool with
    • param-file “Ranked Genes”: the output of Cut tool
    • param-check “File has header?”: Yes
    • param-file “Gene Sets”: h.all.v7.4.symbols.gmt (this should be tabular format, if not, see how to change it in the Tip below)
    • param-text “Minimum Size of Gene Set”: 15
    • param-check “Output plots”: Yes

    question Questions

    What is the top ranked pathway?

    solution Solution

    Oxidative phosphorylation. You can see some explanation of fgsea output in the RNA-seq genes to pathways” tutorial.

(Optional) Multiple conditions

It takes about 30 mins to run mageck mle on the samples below so this part of the tutorial is optional, you can try it if time allows or in your own time.

If we have more than two conditions to compare, or a complex experimental design, we can use MAGeCK mle. MAGeCK mle uses a maximum likelihood estimation (MLE) algorithm (Li et al. 2015). It outputs a single value (beta score) per gene instead of a score for both negative and positive selection. A negative beta score indicates negative selection and a positive indicates positive selection. MAGeCK mle can also be used for comparing 2 conditions instead of MAGeCK test (RRA) but it is slower.

To demonstrate using MAGeCK mle, we will compare the drug treatment (T8-APR-246) to T0 and the vehicle (T8-Vehicle) to T0. We’ll use a design matrix file as described here. The rules of the design matrix are:

  • The design matrix file must include a header line of condition labels
  • The first column is the sample labels that must match labels in read count file
  • The second column must be a “baseline” column that sets all values to “1”
  • The value in the design matrix is either “0” or “1”
  • You must have at least one sample of “initial state” (e.g., day 0 or plasmid) that has only one “1” in the corresponding row. That only “1” must be in the baseline column
  • The initial state (baseline) sample must be the 1st sample listed, under the header row, for mageck mle

The file for our samples looks like below. We have 3 conditions: baseline (T0), Vehicle and APR. The 1st row indicates that T0 is our baseline sample, the 2nd row indicates that we want to compare Vehicle to T0, and the 3rd row indicates that we want to compare APR to T0.

Samples     baseline  Vehicle APR
T0-Control  1         0       0
T8-Vehicle  1         1       0
T8-APR-246  1         0       1

Examples of more complicated design matrices, for e.g. time series experiments, can be seen on the MAGeCK website.

hands_on Hands-on: Test for enrichment with MLE

  1. Import the design matrix file from Zenodo or the Shared Data library (if available):
    https://zenodo.org/api/files/6599878c-f569-41bf-a37a-2c6f3d2e67f9/kenji_mageck_mle_design_matrix.tsv
    
  2. MAGeCKs mle Tool: toolshed.g2.bx.psu.edu/repos/iuc/mageck_mle/mageck_mle/0.5.9.2.1 with the following parameters:
    • param-file “Counts file”: the kenji_mageck_sgrna_counts.tsv file
    • “Design matrix or sample labels”: Design matrix
      • param-file “Design matrix file”: the mageck_mle_design_matrix file

MAGeCK mle Gene Summary output is described here and below.

Column Content
Gene Gene ID
sgRNA The number of targeting sgRNAs for each gene
Condition_name|beta The beta score of this gene in condition (e.g. Vehicle or APR). The conditions are specified in the design matrix input to mle.
Condition_name|p-value The raw p-value (using permutation) of this gene
Condition_name|fdr The false discovery rate of this gene
Condition_name|z The z-score associated with Wald test
Condition_name|wald-p-value The p value using Wald test
Condition_name|wald-fdr The false discovery rate of the Wald test

Similar to what we did with the MAGeCK test output, we can create a volcano plot to visualise the results, showing the magnitude of change (beta score) and significance (p-value).

hands_on Hands-on: Visualize MLE results

  1. Create a volcano plot to visualise the result for APR vs T0. Use the gene summary file columns Gene APR|beta APR|wald-p-value APR|wald-fdr
    • Volcano Plot Tool: toolshed.g2.bx.psu.edu/repos/iuc/volcanoplot/volcanoplot/0.0.5 to create a volcano plot
      • param-file “Specify an input file”: the MAGeCK mle Gene Summary file
      • param-select “FDR (adjusted P value)”: Column 14
      • param-select “P value (raw)”: Column 13
      • param-select “Log Fold Change”: Column 9
      • param-select “Labels”: Column 1
      • param-select “Points to label”: Significant
      • param-text “Only label top most significant”: 10

Volcano plot for APR compared to timepoint zero.

Similarly, you could create a plot for the vehicle vs T0 using the gene summary file columns Gene Vehicle|beta Vehicle|wald-p-value Vehicle|wald-fdr

tip Tip: Getting help

For questions about using Galaxy, you can ask in the Galaxy help forum. For questions about MAGeCK, you can ask in the MAGeCK Google group.

Conclusion

CRISPR Screen reads can be assessed for quality using standard sequencing tools such as FASTQC, MultiQC and trimmed of adapters using Cutadapt. The detection of enriched guides can be performed using MAGeCK. Downstream analysis can include visualisations, such as volcano plot, and pathway analysis with tools like fgsea.

Acknowledgements

Thanks to Mehmet Tekman for suggesting the awk tool. Thanks also to Jennifer Devlin, Lydia Lim and Sylvia Mahara for comments and feedback on the tutorial.

Key points

  • CRISPR screen data can be analysed using MAGeCK and standard read quality tools

Frequently Asked Questions

Have questions about this tutorial? Check out the tutorial FAQ page or the FAQ page for the Genome Annotation topic to see if your question is listed there. If not, please ask your question on the GTN Gitter Channel or the Galaxy Help Forum

References

  1. Marcel, M., 2011 Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17: 10–12. http://journal.embnet.org/index.php/embnetjournal/article/view/200
  2. Li, W., H. Xu, T. Xiao, L. Cong, M. I. Love et al., 2014 MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. 15: 10.1186/s13059-014-0554-4
  3. Li, W., J. Köster, H. Xu, C.-H. Chen, T. Xiao et al., 2015 Quality control, modeling, and visualization of CRISPR screens with MAGeCK-VISPR. 16: 10.1186/s13059-015-0843-6
  4. Doench, J. G., N. Fusi, M. Sullender, M. Hegde, E. W. Vaimberg et al., 2016 Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nature Biotechnology 34: 184–191. 10.1038/nbt.3437
  5. Ewels, P., M. Magnusson, S. Lundin, and M. Käller, 2016 MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics 32: 3047–3048. https://academic.oup.com/bioinformatics/article/32/19/3047/2196507
  6. Joung, J., S. Konermann, J. S. Gootenberg, O. O. Abudayyeh, R. J. Platt et al., 2016 Protocol: Genome-scale CRISPR-Cas9 Knockout and Transcriptional Activation Screening. 10.1101/059626
  7. Chen, C.-H., T. Xiao, H. Xu, P. Jiang, C. A. Meyer et al., 2018 Improved design and analysis of CRISPR knockout screens (J. Wren, Ed.). Bioinformatics 34: 4095–4101. 10.1093/bioinformatics/bty450
  8. Cluse, L. A., I. Nikolic, D. Knight, P. B. Madhamshettiwar, J. Luu et al., 2018 A Comprehensive Protocol Resource for Performing Pooled shRNA and CRISPR Screens, pp. 201–227 in Methods in Molecular Biology, Springer New York. 10.1007/978-1-4939-7568-6_17
  9. Fujihara, K. M., B. Zhang, T. D. Jackson, B. Nijiagel, C.-S. Ang et al., 2020 Genome-wide CRISPR screens reveal APR-246 (Eprenetapopt) triggers ferroptosis and inhibits iron-sulfur cluster biogenesis. 10.1101/2020.11.29.398867
  10. Przybyla, L., and L. A. Gilbert, 2021 A new era in functional genomics screens. Nature Reviews Genetics. 10.1038/s41576-021-00409-w
  11. Uyhazi, K. E., and J. Bennett, 2021 A CRISPR view of the 2020 Nobel Prize in Chemistry. Journal of Clinical Investigation 131: 10.1172/jci145214
  12. Bock, C., P. Datlinger, F. Chardon, M. A. Coelho, M. B. Dong et al., 2022 High-content CRISPR screening. Nature Reviews Methods Primers 2: 10.1038/s43586-021-00093-4

Glossary

CRISPR
Clustered Regularly Interspaced Short Palindromic Repeats
MAGeCK
Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout

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Citing this Tutorial

  1. Maria Doyle, Kenji Fujihara, Twishi Gulati, 2022 CRISPR screen analysis (Galaxy Training Materials). https://training.galaxyproject.org/training-material/topics/genome-annotation/tutorials/crispr-screen/tutorial.html Online; accessed TODAY
  2. Batut et al., 2018 Community-Driven Data Analysis Training for Biology Cell Systems 10.1016/j.cels.2018.05.012

details BibTeX

@misc{genome-annotation-crispr-screen,
author = "Maria Doyle and Kenji Fujihara and Twishi Gulati",
title = "CRISPR screen analysis (Galaxy Training Materials)",
year = "2022",
month = "03",
day = "09"
url = "\url{https://training.galaxyproject.org/training-material/topics/genome-annotation/tutorials/crispr-screen/tutorial.html}",
note = "[Online; accessed TODAY]"
}
@article{Batut_2018,
    doi = {10.1016/j.cels.2018.05.012},
    url = {https://doi.org/10.1016%2Fj.cels.2018.05.012},
    year = 2018,
    month = {jun},
    publisher = {Elsevier {BV}},
    volume = {6},
    number = {6},
    pages = {752--758.e1},
    author = {B{\'{e}}r{\'{e}}nice Batut and Saskia Hiltemann and Andrea Bagnacani and Dannon Baker and Vivek Bhardwaj and Clemens Blank and Anthony Bretaudeau and Loraine Brillet-Gu{\'{e}}guen and Martin {\v{C}}ech and John Chilton and Dave Clements and Olivia Doppelt-Azeroual and Anika Erxleben and Mallory Ann Freeberg and Simon Gladman and Youri Hoogstrate and Hans-Rudolf Hotz and Torsten Houwaart and Pratik Jagtap and Delphine Larivi{\`{e}}re and Gildas Le Corguill{\'{e}} and Thomas Manke and Fabien Mareuil and Fidel Ram{\'{\i}}rez and Devon Ryan and Florian Christoph Sigloch and Nicola Soranzo and Joachim Wolff and Pavankumar Videm and Markus Wolfien and Aisanjiang Wubuli and Dilmurat Yusuf and James Taylor and Rolf Backofen and Anton Nekrutenko and Björn Grüning},
    title = {Community-Driven Data Analysis Training for Biology},
    journal = {Cell Systems}
}
                

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