CRISPR-OFF webserver (v1.1): off-targeting assessment of Cas9 gRNAs

This webserver is dedicated to the off-targeting assessment of different gRNA/on-target selections for CRISPR-Cas9 applications. Using the webserver, you can compute the CRISPRoff off-target confidence scores of off-target predictions of a gRNA and/or you can compare different gRNA/on-target selections using the CRISPRspec specificity score.

Format for INPUT-1: gRNA/target sequence(s)

You can either add your gRNA sequence(s) or the target sequence(s) that you would like to design gRNAs for.

Example 1 (single gRNA sequence):

Example 2 (multiple gRNA sequences in fasta format):

Example 3 (a target sequence):

Example 4 (multiple target sequences in fasta format):

Input criteria

(1) All given sequences must only contain the following five canonical bases 'A,a', 'C,c', 'G,g', 'T,t' and 'U,u'.
(2) Given sequence(s) must not exceed 2300nts in total and must not contain more than 100 gRNAs.
(3) Given sequence(s) cannot be shorter than 23nts.
For those inputs not stastifying the above criteria, they will not be processed by the CRISPR-OFF webserver..

Good to know!

(1) If you want to upload only a single gRNA (Example 1), it must be 23nts long and have a valid PAM sequence (NGG).
(2) gRNAs with invalid PAM sequence (other than NGG) will always be discarded.
(3) You can upload your gRNA/target sequences in fasta format as well (Example 2, 4).
(4) Sequences longer than 23nts will always be treated as target sequences and potential gRNAs with NGG PAM on this target (including the reverse strand) will be generated by the webserver.

Format for INPUT-2: off-target predictions

To perform the off-target predictions within our webserver, simply choose your organism from the dropdown menu. If desired organism is not part of our webserver, you can always upload your own off-target predictions.

Example for user-given off-target predictions:

Input criteria

(1) The input must only contain the following five canonical bases 'A,a', 'C,c', 'G,g', 'T,t' and 'U,u'.
(2) The input must be shorter than 1000000 nts.
(3) Input off-target sequences must be 23nts long, therefore cannot include bulged off-targets.
(4) Given off-target sequences must be seperated with newline.
(5) Input off-target sequences must include the on-target sequence as well.
(6) For more accurate CRISPRspec score computation, DO NOT exclude or filter the repeated off-target sequences.
For those inputs not stastifying the above criteria, they will either not be processed by CRISPR-OFF webserver or computed CRISPRspec and CRISPRoff scores will be inaccurate.

Email and custom job name

It is necessary that you give an email address and an optional custom job name so that we can inform you by email when the computation for your job finishes. You can use the given job ID to access the results for your job any time later or simply wait until the computation is done (should take around 10mins, but also might take over an hour when server is busy). The Results page will be automatically updated every ten seconds until the computation for the job has finished.
Please provide a valid e-mail address.


Please read the About page for more details on results.

CRISPRoff score and off-target predictions

When comparing different off-target predictions, higher the CRISPRoff score gets it hints to higher confidence on the cleavage of the putative off-target.

CRISPRspec score and off-targeting potential

When comparing the specificity of the different gRNA off-target predictions, higher the CRISPRspec score gets it hints to higher specificity and lower off-targeting potential for that gRNA.


CRISPR-Cas9 off-targeting assessment with nucleic acid duplex energy parameters
Alkan F, Wenzel A, Anthon C, Havgaard JH, Gorodkin J Genome Biol. 2018 Oct 26;19(1):177
[ PubMed | Paper | Webserver | Software ]


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