Computational pipeline for RNA-seq based on-target and off-target assessment of Cas9-mediated edits


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 can 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), making it the first comprehensive pipeline for the analysis of RNA-seq data from CRISPR/Cas9 edited cells and controls.



The CRISPRroots pipeline is written as a Snakefile which can be executed via Snakemake. We recommend snakemake installation via conda so that `--use-conda` flag could be used while calling snakemake to let it automatically handle all dependencies of the pipeline.
  • conda : ≥4.8.5
  • snakemake : ≥5.32.0

Get software and test dataset

You can download the CRISPRroots software and the test dataset here.


After downloading, unpack and follow the instructions given in the README.html or file.
tar -xzvf CRISPRroots-1.0.tar.gz
cd CRISPRroots-1.0/CRISPRroots
firefox README.html

Assuming you have installed the software in the prerequisites, and have downloaded, un-packed and setup the `config.yaml` present in the test sample directory (see README), you can run the pipeline for the sample data from the sample directory using the following command:
snakemake -s [path_to_CRISPRroots]/run.smk --use-conda --dry-run
snakemake -s [path_to_CRISPRroots]/run.smk --use-conda
We recommend to first run Snakemake with `--dryrun`. This displays what will be done without executing it and highlights if any input file is missing. If there are no error messages then CRISPRroots is setup correctly and ready to be launched (without `--dryrun`)


  • For full details about how to use the CRISPRroots software, 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. Submitted.


In case of problems or bug reports, please contact