CRISPRroots: CRISPR–Cas9-mediated edits with accompanying RNA-seq data assessed for on-target and off-target sites.


The CRISPR/Cas9 genome editing tool can be used to study genomic variants and gene knockouts. By combining CRISPR/Cas9 mediated editing with transcriptomic analyses it is possible to measure the effects of genome alterations on gene expression. In such experiments it is crucial to understand not only if the editing was successful but also if the observed differential gene expression is the result of intended genome edits and not that of unwanted off-target effects. Potential off-targets sites that CRISPR/Cas9 may have edited need therefore to be verified by sequencing. However, a CRISPR/Cas9 gRNA may have hundreds (or thousands) potential off-target binding sites in a genome. How to decide which of them should be validated with higher priority? The RNA-seq data that may be sequenced as part of a CRISPR/Cas9 experiment contain information about the sequence and expression level of potential off-target sites/genes located in transcribed regions. The CRISPRroots pipeline implements a method that combines CRISPR/Cas9 and guide RNA binding properties, gene expression changes, and sequence variants between edited and non-edited cells, to discover and rank potential off-targets. Additionally, CRISPRroots verifies on-target editing changes and performs several bioinformatic analyses on the RNA-seq data (eg. gene differential expression). This makes CRISPRroots the first comprehensive pipeline for the analysis of RNA-seq data from CRISPR/Cas9 edited cells and controls.



CRISPRroots is a Snakemake pipeline. A docker container including all the required tools to run the pipeline is available on DockerHub. Therefore, to run the pipeline the only requirements are Snakemake itself and Apptainer for singularity support.
  • snakemake : ≥7.28.3
  • apptainer : ≥1.1.8

Get software and test dataset

You can download the CRISPRroots software and the test dataset here:
Previous versions of the CRISPRroots software and the test dataset are available here:
CRISPRroots is also available on GitHub here.


After downloading, unpack and follow the instructions given in the file.
tar -xzvf CRISPRroots-1.2.tar.gz
cd CRISPRroots-1.2/CRISPRroots
After having installed Snakemake and apptainer and having prepared your singularity of the CRISPRoots container, you need to setup the `config.yaml` before running the pipeline. An example of the config file is given in the CRISPRroots folder under the subfolder `resources`, as well as in the test directory. Once the config is ready, you can run the pipeline as follows:
snakemake -s [path_to_CRISPRroots]/run.smk --cores  --use-singularity --singularity-args 
"--bind [global_path_to_QPRT_DEL268T_chr16_10M-40M]:[global_path_to_QPRT_DEL268T_chr16_10M-40M]
--bind [global_path_to_resources]:[global_path_to_resources]
--bind [global_path_to_CRISPRroots-1.3]:[global_path_to_CRISPRroots-1.3]"
The number of --bind options and their paths depend on where the data required by the pipeline is stored. The --bind options are required to link your local files to the file system within the CRISPRroots container. We recommend to first run this command with option `--dryrun`. This displays what will be done without executing it and highlights if any input file is missing. If there are no error message then CRISPRroots is setup correctly and ready to run (without `--dryrun`).


  • For more details on how to use CRISPRroots, please read the CRISPRroots_Manual.pdf distributed with the software package.


When using this software, please cite:
CRISPRroots: on- and off-target assessment of RNA-seq data in CRISPR-Cas9 edited cells
Corsi GI, Gadekar VP, Gorodkin J, Seemann SE. Nucleic Acids Research (2021).


In case of problems or bug reports, please contact