#CRISPR Library Designer (CLD): a software for the multispecies design of sgRNA libraries
ABSTRACT
Genetic screens using CRISPR/Cas9 are a powerful method for the functional analysis of genomes. Here we provide a fully integrated bioinformatics workflow for the design of custom single guide (sg) RNA libraries for a broad spectrum of organisms, termed CRISPR library designer (CLD). CLD can predict a high fraction of functional sgRNAs. An analysis on parameters that determine on-target efficiency of sgRNAs indicates that their prediction by CLD gives valuable insights into their efficiency in experiment. CLD enables the design of custom scalable, high-coverage sgRNA libraries for many species.
Quick-Start:
On Mac: If you haven't, install Xquartz from http://www.xquartz.org/
When logging in remotely: log into your remote server by ssh -X
Download CLD_GUI_Mac.zip or CLD_GUI_Ubuntu.zip according to your Operating system and unzip it.
Double click on the application or open it by ./CLD in the Terminal.
Download the database for your organism of interest.
Enter its name in the reference organism field on the start page.
Enter a list of gene identifiers in the "Gene List" tab and go to the "Design Parameter" tab to set your parameters.
Go to the "Start Analysis" tab to start sgRNA search.
The results will be created in the selected output directory ("Input/Output" tab).
Command-Line-Start:
cld can be called either with “--version”, printing its version number and copyrights, “--help” printing a more elusive help documentation and with “--task”.
EXAMPLE to execute from the path containing all needed files:
cld -—task=end_to_end —-output-dir=. --parameter-file=./params.txt --gene-list=./gene_list.txt
cld can run 2 distinct tasks, database creation and library design.
Database creation is called using the “--task=make_database” command giving the organism name of interest, as it is denoted in ENSEMBLs ftp folder structure e.g. homo_sapiens, and the rsync url to the current ftp server of ENSEMBL, examples can be found when cld --help is called. After calling this function CLD will automatically download the latest toplevel FASTA, GFF and GTF files for the organism of interest and compile a database containing bowtie indexes, mygff files and reformatted sequence files. If not enough computing power is available to the user, these databases also might be downloaded from http://www.dkfz.de/signaling/crispr-downloads/.
Library design can either be done in two steps: “cld --task=target_ident” and then “cld --task=library_assembly” if the user wants to separate the two steps for example in order to only identify target sites without compiling a clonable library. Else “cld --task=end_to_end” which automatically will perform the steps mentioned before and present the end-result in a user defined output folder. For reasons of manageability for high throughput design, output files are kept as simple and standardised as possible. However a genome wide library targeting the human genome quickly spans several GB depending on how strict the parameters are chosen. Since the end_to_end task takes most time we benchmarked its time consumption to be approximately 1 h wall-time for an 8-core cpu node.
For running cld from the command line the syntax as outlined in the MANUAL must be used.