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CasCADe: a fully integrated and open source pipeline to design CRISPR mutagenesis experiments
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README.md

CasCADe: a fully integrated and open source pipeline to design CRISPR mutagenesis experiments

Contents

Hardware Requirements
Installing With Docker
Installing Natively
Customization
Migration
Troubleshooting

Hardware Requirements

CasCADe can be installed on any machine running a docker-compatible operating system. Alternately, to run CasCADe natively, Ubuntu 16.04 is required.

In either case, a minimum of 8GB of RAM and 100GB of hard disk space is recommended.

Installing with Docker

First, install the docker engine. If using Linux, also install docker compose (this is included by default in Mac and Windows installations of docker).

Clone the CasCADe repository:

    git clone https://github.com/ccmbioinfo/CasCADe.git

Navigate to the CasCADe directory. By default, the .env file defines yeast (Saccharomyces_cerevisiae) as the organism of interest with genome assembly R64-1-1:

    ORGANISM=Saccharomyces_cerevisiae
    ASSEMBLY=R64-1-1

Change these defaults to your desired organism/genome build if they are different. A full list of available genomes can be found at ftp://ftp.ensembl.org/pub/release-98/fasta.

From within the CasCADe directory, build the docker container:

docker-compose up

Depending on the size of your organism, it may take several hours to download the genome and build the index files. Once the setup is complete, the message: * Starting Apache httpd web server apache2 * will be displayed. CasCADe should be available via web browser at your server's domain address (or at localhost if running locally).

Installing Natively

(Requires sudo privileges and a machine running Ubuntu 16.04)

Install git and apache2:

  sudo apt-get update
  sudo apt-get install -y git apache2

Navigate to a folder where you would like to install CasCADe. It must be a folder that can be served by apache2 (i.e. var/www/html) and remove the index.html file if it exists

cd /var/www/html
rm index.html

Clone the CasCADe repo:

  git clone https://github.com/ccmbioinfo/CasCADe.git

Then, run the installation script using the command below

   cd CasCADe/src/installation
   ./install.sh /var/www/html/CasCADe

If the installation process halts with a message Default Kerberos version 5 realm:, press Enter

After the installation step is completed, start the mongodb server: systemctl start mongod

Then, navigate to the src/setup folder under the root directory (e.g./var/www/html/CasCADe/src/setup), and run the following command to setup Jbrowse, the BLAST database and mongodb.

    python3 setup.py -r /var/www/html/CasCADe -g Saccharomyces_cerevisiae -v R64-1-1 -fa2bit /var/www/html/CasCADe/bin/faToTwoBit -b /var/www/html/CasCADe/bin/ncbi-blast-2.7.1+/bin

Replace the default organism (Saccharomyces_cerevisiae) and its genome build (R64-1-1) with your organism and build of interest. A full list of available genomes can be found at ftp://ftp.ensembl.org/pub/release-98/fasta.

Now, run the script to grant apache the correct file permissions.

    cd /var/www/html/CasCADe/src/installation
    ./setPermissions.sh

Next, the apache server needs to be configured to make the tool web accessible.

Open the file /etc/apache2/sites-available/000-default.conf, modify the DocumentRoot to the location where CasCADe was installed, and add this section below:

    DocumentRoot /var/www/html/CasCADe
    <Directory '/var/www/html/CasCADe'>
        Options +ExecCGI
        AddHandler cgi-script .py
        Allow from all
    </Directory>

Then, restart apache2

    systemctl restart apache2

CasCADe should now be accessible from your browser at http://<your servers address>. For example, if your VM's IP address is 172.20.20.20, you can type : http://172.20.20.20 in your browser to access CasCADe.

To enable In Silico PCR tool Dicey (a.k.a. Silica), run the enable_dicey.sh script. Again provide your organism of interest and genome assembly in place of Saccharomyces_cerevisiae and R64-1-1:

 cd /var/www/html/CasCADe/src/setup
 ./enable_dicey Saccharomyces_cerevisiae R64-1-1

Which will fetch and name the index files if they're available.

If you want your installation of CasCADe to be secured by https, please refer to apache2's documentation on how to enable https module.

Customization

Custom Primer Design Settings

First, go to to the primer3 website and enter the custom settings that you would like to use for the first attempt of primer design and click 'Download Settings' to save the file. Additionally, you may also specify and download 'retry attempt' settings to be used if no primers are found with the default settings. There is no limit to the number of retry attempts you can define.

Once all the settings files have been generated, ssh into the server hosting CasCADe and navigate to where the application is rooted (e.g. /var/www/html/CasCADe). Within the config directory there should be a primer3settings.conf file and a directory, primer3settings, where the default primer3 settings are stored. Replace these with your custom settings and edit the primer3settings.conf file to point to the new files like so:

ATTEMPT_0_FILE=primer3settings/filename.txt
ATTEMPT_1_FILE=primer3settings/filename1.txt
ATTEMPT_1_DESC="Description of changes in ATTEMPT_1 file"
ATTEMPT_2_FILE=primer3settings/filename2.txt
ATTEMPT_2_DESC="Description of changes in ATTEMPT_2 file"

The descriptions are optional but will be displayed to users if a design failure occurs.

Custom RNA-guided Endonuclease (RGEN) Settings

The src/setup/rgens.json defines the default RGEN settings:

Shortform PAM PamLocation MinGuideLength MaxGuideLength DefaultGuideLength SeedRegion Cleaves OffTargetPAMs Scores
SpCas9 (NGG) NGG downstream 17 20 20 -12 -3 NGG, NAG MIT, CFD
AsCpf1/Cas12a TTTV upstream 20 23 - +6 +19,+23 TTTV, TTTT, CTTA, TTCA -
ScCas9 (NNG) NNG downstream 20 20 - -12 -3 NNG -
ScCas9 (NNGT) NNGT downstream 20 20 - -12 -3 NNGT -
SaCas9 NNGRRT downstream 21 23 - -8 -3 NNGRRT, NNGRR -

However, we recognize that these settings and defaults may not be applicable to all users. New RGENs can be added by specifying the RGEN parameters in the template below and appending to the rgens.json file.

{"rgenID" : "6", "Shortform" : "<shortform>", "Longform" : "<optional>", "PAM" : "<nucleotide sequence>", "PamLocation" : "<downstream or upstream>", "MinGuideLength" : "<required>", "MaxGuideLength" : "<required>", "DefaultGuideLength": "<optional>", "SeedRegion" : "<direction and length of seed>", "Cleaves" : [ "<cleavage site>" ], "OffTargetPAMS" : [ "<actual PAM>", "<optional off-targets>" ], "Scores" : []}

Existing RGENs can have their settings and defaults modified in the file as well. In order to keep existing guides associated to their RGENs, it's recommended that the rgenIDs remain unchanged for existing entries.

After modifying the rgens.json file, you can run the load_RGENs.py script in the same directory (python3 load_RGENS.py) and provide the desired command-line argument:

  • update: modifies existing records based on the rgenID and adds new RGENs to the database (recommended)
  • replace: wipes and replaces RGEN database with new entries, potentially unlinking existing gRNA records from their RGEN.

Migration

If using a previous version of CasCADe, existing Primers and Guides can be migrated to the new version by performing the following steps:

  1. Navigate to the existing CasCADe installation and make copies of the collections to migrate. Replace mm10 with your genome version, if it is different, and define a directory where the database files should be output.
 mongodump --db=mm10 --collection=gRNAResultCollection -o <output_directory>
 mongodump --db=mm10 --collection=primerCollection -o <output_directory>

This will place the .bson and .json files into a folder in your specified output directory. Transfer this folder to the new CasCADe installation if it is on a different machine.

  1. On the server hosting the new CasCADe, run:
 mongorestore --db=mm10 --collection gRNAResultCollection <path_to_transferred_folder>/gRNAResultCollection.bson
 mongorestore --db=mm10 --collection primerCollection <path_to_transferred_folder>/primerCollection.bson
  1. After restoring the database, the database documents need to be converted to the new format via a Python script:
cd /var/www/html/CasCADe
sudo python3 customPython/MongoConverter.py
  1. Finally, the GFF files need to be re-written for the JBrowse tracks by a python script:
sudo python customPython/MongoHandler.py mm10

Your existing Primer and Guide designs should now be accessible in the new CasCADe installation!

Troubleshooting

If you encounter issues installing or using CasCADe, raise a GitHub Issue or send an email to hillary(dot)elrick(at)sickkids(dot)ca

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