CRISPR-offinder: A program used for designing CRISPR guide RNA and searching off-targets for user-defined protospacer adjacent motif (PAM)

CRISPR-offinder requires an OpenCL-enabled device (CPU, GPU, or etc..) and corresponding runtime driver pre-installed to run properly.

Please download and install the latest driver below:
1.Intel (Download 'OpenCL runtime' in the middle of the page):
4.Perl; Python >=2.7 or greater
5.Python package: Scipy,Biopython and Sci-kit learn, you can install by commond:
   sudo pip install BioPython
   sudo pip install sklearn
   sudo pip install scipy

Before installing CRISPR-offinder, please check whether your device is an OpenCL-supported one.

Downloads: ‍


      cd CRISPR-offinder_1.2

Supported OS
1.Linux (with proprietary drivers installed)
2.Mac OS X (Snow leopard or higher)

        Version: 1.2
        Feb 24, 2016

        $ perl ./ <option>
        For help information, type 'perl'

Given an input FASTA file of the target sites and queries the reference genome as well as a CRISPR system with a defined spacer length and PAM sequence, this standalone tool will identify putative sites and assign a predicted activity based on support vector machine model which conducted by sgRNA Scorer 2.0. In addition, sgRNAs with minimal off-target activity were predicted by Cas-OFFinder, and score with Off-Target Cutting Frequency Determination (CFD).

        Free for research and educational users. version 1.2
 perl <option>
    -input           [s] Input file in FASTA format <required>
   -pamseq        [s] PAM sequence <required>
   -pamori         [s] PAM orientation. Value must either be 5'or 3' (enter 5 or 3)  <deafult: 3>
   -pamlen        [i] Length of protospacer <default: 20>
   -gc_min        [i] The minimum value of GC content <default: 20>
   -gc_max       [i] The maximum value of GC content <default: 80>
   -mismatches [i] Number of mismatches[0-9] <default: 5>
   -strand          [s] Searching CRISPR target sites using DNA strands based option(s/a/b) <default: b>
   -cga               [s] (C: using CPUs, G: using GPUs, A: using accelerators) <default: C>
   -gd                [s] genome dir <default: $Bin/genome>
   -system         [s] run system (Linux32/Linux64/Mac) <default: Linux64>
   -offset_start  [i] The minimum value of sgRNA offset <default: -2>
   -offset_end   [i] The maximum value of sgRNA offset <default: 32>
   -output          [s] Output dir. Final output files with guide RNA sequences and scores

<default: ./>
Whole genome of target organism is needed (in FASTA format). You can find one in one of the below links:
1.UCSC genome sequences library,

2.Ensembl sequence library,

Extract all FASTA files in a directory. Remember the full path of the FASTA files directory(for option -gd).

For help information: perl

PAM requirement:
   NGG - SpCas9 from Streptococcus pyogenes - direction: 3’
   NRG - SpCas9 from Streptococcus pyogenes - direction: 3’
   NNAGAAW - StCas9 from Streptococcus thermophilus - direction: 3’
   NNNNGMTT - NmCas9 from Neisseria meningitidis - direction: 3’
   NNGRRT - SaCas9 from Staphylococcus aureus - direction: 3’
   NNNRRT - SaCas9 KKH variant - direction: 3’
   NGG(reduced NAG binding) - SpCas9 D1135E variant - direction: 3’
   NGCG - SpCas9 VRER variant - direction: 3’
   NGAG - SpCas9 EQR variant - direction: 3’
   NGAN-NGNG - SpCas9 VQR variant - direction: 3’
   NGG - FnCas9 from Francisella novicida - direction: 3’
   YG - FnCas9 RHA variant - direction: 3’
   TTTN - AsCpf1 from Acidaminococcus, LbCpf1 from Lachnospiraceae - direction: 5’
   TTN - FnCpf1 from Francisella novicida strain U112 - direction: 5’
   CTA - FnCpf1 from Francisella novicida strain U112 - direction: 5’
   TTN-CTA - FnCpf1 from Francisella novicida strain U112 - direction: 5’
   TTN - C2c1 from four major taxa: Bacilli, Verrucomicrobia, a-proteobacteria, and d-proteobacteria - direction: 5’
   Custom, Enter your PAM - enter user defined-PAM

   Code  Base       Code  Base
   A     Adenine     K      G or T
   C     Cytosine     M     A or C
   G     Guanine     B      C or G or T
   T     Thymine     D     A or G or T
   R     A or G        H     A or C or T
   Y     C or T        V     A or C or G
   S     G or C         N    any base
   W    A or T      
Note that CRISPR-offinder allows mixed bases to account for the degeneracy in PAM sequences

CRISPR-offinder Frequently Asked Questions

1. What is CRISPR-offinder?

CRISPR-offinder is a CRISPR sgRNA design and off-target searching tool for user-defined protospacer adjacent motif (PAM). This tool could help predict off-target activity and design highly specific sgRNA for use in clinical and agricultural applications of CRISPR system.

2. What is a PAM sequence and where is it located?

CRISPR-Cas9/Cpf1/C2c1 mechanisms recognize DNA targets that are complementary to a short CRISPR sgRNA sequence. The part of the sgRNA sequence that is complementary to the target sequence is known as a protospacer. In order for Cas9/Cpf1/C2c1 to function it also requires a specific protospacer adjacent motif (PAM) that varies depending on the bacterial species of the Cas9/Cpf1/C2c1 gene.

Recognition of the PAM by the Cas9/Cpf1/C2c1 nuclease is thought to destabilize the adjacent sequence, allowing interrogation of the sequence by the sgRNA, and resulting in RNA-DNA pairing when a matching sequence is present. Cas9 nucleases with alternative PAMs have also been characterized and successfully used for genome editing. It is important to note that the PAM is not present in the sgRNA sequence but needs to be immediately downstream or upstream of the target site in the genomic DNA.

3. What is the CRISPR/Cas9 system?

Cas9/gRNA system, one type of the CRISPR/Cas systems, is a kind of engineered endonuclease (Fig1). It consists of two components: Cas9, a protein with DNA nuclease activity can be used universally in this system; and sgRNA, an ~100-nt single guide-RNA, of which the first ~20 nt in the 5'-end is responsible for recognizing the target site DNA in a DNA-RNA complementary manner. Cas9/gRNA recognizes and cleaves the target DNA and causes a DSB (double-strand break), which provides the opportunity of gene mutagenesis and other types of genome manipulation.

Fig1. The CRISPR/Cas9 system for targeted genome editing

4. What is the CRISPR/Cpf1 system?

CRISPR/Cpf1 is a DNA-editing technology (Fig2). It works analogously to CRISPR/Cas9 which has revolutionized biological research. Like its predecessor, it is derived from a mechanism that bacteria use to prevent genetic damage from viruses. CRISPR/Cpf1 may be better than CRISPR/Cas9 in that Cpf1 is a smaller and simpler endonuclease (a type of enzyme) than Cas9. That simplifies delivery to the cells whose genes need modifying. Two candidate enzymes from Acidaminococcus and Lachnospiraceae display efficient genome-editing activity in human cells.

Fig2. The CRISPR/Cpf1 system for targeted genome editing

5. What is the CRISPR/C2c1 system?

CRISPR/C2c1 is a DNA-editing technology (Fig3). It works analogously to CRISPR/Cpf1 which has revolutionized biological research. C2c1 system can mediate DNA interference in a 5’-PAM-dependent fashion analogous to Cpf1. However, unlike Cpf1, which is a single-RNA-guided nuclease, C2c1 depends on both crRNA and tracrRNA for DNA cleavage.

Fig3. The CRISPR/C2c1 system for targeted genome editing

6. What is the Paired-gRNAs strategy of CRISPR TECHNOLOGY?

The paired-gRNAs strategy is reported to show higher specificity than the above single-gRNA strategy, it consists of three components: Cas9-nickase, a mutant form of Cas9 protein, which has no nuclease activity but nickase activity, only cleaving one of the DNA strands with the assist of one gRNA; and a pair of gRNAs, target two sites with offset no more than tens of nt in the opposite strands of DNA (Fig4). The two close nicks induced by the gRNA pair can cause a DSB. However, single nick in a potential off-target induced by only one gRNA would be difficult to cause DSB.

Fig4. The Paired-gRNAs strategy of CRISPR TECHNOLOGY

7. What is the potential off-target site?

Potential off-target site is an unwanted target site in the genome. This is an important consideration when application of CRISPR technology. The specificity of the CRISPR system is determined in large part by how specific the sgRNA targeting sequence is for the genomic target compared to the rest of the genome. Ideally, a sgRNA will have perfect match to the target DNA with no homology elsewhere in the genome. In fact, most of sgRNA targeting sequence will have additional sites throughout the genome where partial homology exists.

8. Is the PAM sequence part of the sgRNA sequence construct?

The PAM sequence is located on the non-complementary strand. In other words, it is on the strand of DNA that contains the same DNA sequence as the target sgRNA. The PAM sequence should not be included in the design of the sgRNA.

For citation:

Zhao C, Zheng X, Qu W, Li G, Li X, Miao Y, Han X, Liu X, Li Z, Ma Y, Shao Q, Li H, Sun F*, Xie S*, Zhao S*. CRISPR-offinder: a CRISPR guide RNA design and  off-target searching tool for user-defined protospacer adjacent motif. Int J Biol Sci, 2017,13(11):1470-1478.‍d‍oi:10.7150/ijbs.21312‍.

For commercial use, please contact the author.

Please send bug reports to:ssxie\

Copyright © 2016, Huazhong Agricultural University.