by Jan Grau, Jens Boch, and Stefan Posch.
CRISPRer is a tool for genome-wide selection and assessment of CRISPR/Cas protospacers. CRISPRer offers three distinct tasks. First, CRISPRer may search for protospacers with unique seed sequences, i.e., with seed sequences which occur only once in a given input genome. Second, it may search for seed sequences that are orthogonal to the input genome, i.e., which do not occur in the given input genome. Third, it may assess a set of user-provided protospacers by collecting several statistics about genome-wide perfect and imperfect matches to their seed sequences.
We provide CRISPRer as a public web-server, a web-application that can be installed in a local Galaxy server, and as a command line program.
CRISPRer is available as a public web-server at galaxy.informatik.uni-halle.de.
CRISPRer is implemented in Java using Jstacs. You can download the command line application as a Jar. In addition, we provide the Jar of the Galaxy web-application for installing it in your local Galaxy server.
CRISPRer will be part of the next public release of the Jstacs library. As (future) part of Jstacs, CRISPRer will be released under GPL 3.
Running the command line application
For running the command line application, Java v1.6 or later is required.
The arguments of the command line application have the following meaning:
|input||Input data (The genome for that CRISPR/Cas protospacers/seeds are selected, FastA format)||String|
|length||Length (The length of the protospacer, default = 20)||Integer|
|seed||Seed length (The length of the seed at the 3' end of the protospacer, valid range = [1, 13], default = 12)||Integer|
|pam||PAM (The PAM pattern, all IUPAC DNA symbols allowed, default = NGG)||String|
|filter||Chromosomal locations (The chromosomal locations for which to filter the reported protospacers. Enter different location separated by semicolons. Ignored if parameter proto set., OPTIONAL)||String|
|proto||Protospacers (Protospacers (without PAM sequence) to assess genome-wide, FastA format, OPTIONAL)||String|
|unique||Unique seeds (If true, CRISPRer searches for protospacers with unique seeds. Otherwise, it searches for orthogonal seeds., default = true)||Boolean|
|include||Include PAM (Include sequence matching PAM pattern in output, default = true)||Boolean|
|prefix||Output prefix (Common prefix of all output files, may include path, default = ./CRISPRer)||String|
|gff2||Use GFF2 (Use GFF2 output format instead of GFF3, default = false)||Boolean|
For instance, using the input parameters
java -Xms512M -Xmx3G -jar CRISPRer.jar input=path/to/genome.fa length=20 seed=12
CRISPRer searches for protospacers of length 20 with unique seeds of length 12 in the genome given in the FastA file
path/to/genome.fa using PAM
NGG and appends the sequence matching the PAM to the output sequences.
If the input parameters are
java -Xms512M -Xmx3G -jar CRISPRer.jar input=path/to/genome.fa seed=10 pam=NCC unique=false include=false
CRISPRer searches for orthogonal seeds of length 10 using PAM
Finally, if the input parameters are
java -Xms512M -Xmx3G -jar CRISPRer.jar input=path/to/genome.fa seed=10 pam=NGG proto=path/to/protospacers.fa
CRISPRer assesses the protospacer sequences given in the FastA file
path/to/protospacers.fa using PAM
NGG and seed length 10.
Installing the web-application
The command-line program behind the web-application is a Jar as well, so Java is required on the server running Galaxy.
To install this command line program in Galaxy, copy it to the desired destination in the Galaxy
The command line application writes its Galaxy tool definition file itself. If you are in the directory containing the command-line program for Galaxy, you can create the tool definition file by calling
java -jar CRISPRerWeb --create CRISPRerWeb.xml
Afterwards, this directory contains the tool definition file
CRISPRerWeb.xml. Now you can register CRISPRer in the Galaxy
tool_conf.xml file. For details, see the Galaxy tutorial for adding new tools.