Bioinformatics Advance Access published December 9, 2014
Bioinformatics, 2014, 1–4
doi: 10.1093/bioinformatics/btu743
Advance Access Publication Date: 20 November 2014
Applications Note

Genome analysis

CRISPRdirect: software for designing CRISPR/
Cas guide RNA with reduced off-target sites
Yuki Naito1,2,*,†, Kimihiro Hino3,†, Hidemasa Bono1,2 and Kumiko Ui-Tei3
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Database Center for Life Science (DBCLS), 2National Institute of Genetics, Research Organization of Informationand Systems, 1111 Yata, Mishima, Shizuoka 411-8540, Japan and 3Department of Biological Sciences, Graduate
School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan*To whom correspondence should be addressed.
†
The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors.
Associate Editor: Alfonso Valencia
Received on May 12, 2014; revised on October 15, 2014; accepted on November 5, 2014

Abstract
Summary: CRISPRdirect is a simple and functional web server for selecting rational CRISPR/Cas
targets from an input sequence. The CRISPR/Cas system is a promising technique for genome engineering which allows target-specific cleavage of genomic DNA guided by Cas9 nuclease in complex with a guide RNA (gRNA), that complementarily binds to a 20 nt targeted sequence. The target sequence requirements are twofold. First, the 50 -NGG protospacer adjacent motif (PAM)
sequence must be located adjacent to the target sequence. Second, the target sequence should bespecific within the entire genome in order to avoid off-target editing. CRISPRdirect enables users toeasily select rational target sequences with minimized off-target sites by performing exhaustive
searches against genomic sequences. The server currently incorporates the genomic sequences ofhuman, mouse, rat, marmoset, pig, chicken, frog, zebrafish, Ciona, fruit fly, silkworm,
Caenorhabditis elegans, Arabidopsis, rice, Sorghum and budding yeast.
Availability: Freely available at http://crispr.dbcls.jp/.
Contact: y-naito@dbcls.rois.ac.jp
Supplementary information: Supplementary data are available at Bioinformatics online.

1 Introduction
Genome engineering is a promising technique to manipulate endogenous chromosomal DNA in a site-specific manner. A novel system that employs the prokaryotic immune defense system based on
the clustered regularly interspaced short palindromic repeats(CRISPR) and CRISPR-associated (Cas) protein has been reported
as a prominent genome engineering approach (Cho et al., 2013;
Cong et al., 2013; Jinek et al., 2013; Mali et al., 2013). Recent studies utilize the RNA-guided endonuclease Cas9 from Streptococcus
pyogenes and a guide RNA (gRNA), which acts as a guide to define
the target site to introduce DNA double-stranded break. A remarkable advantage of the CRISPR/Cas system is that the target DNA sequence is recognized by simple base-pairing complementarity by thegRNA. Thus, the CRISPR/Cas system can be programmed only bychanging the gRNA sequence, and the synthesis of the gRNA fortargeting a specific gene is easy at low cost. However, it should be a
critical issue to avoid the cleavage of the unintended off-target
genes, since double-stranded break results in stable and heritable
modification of the genome.
In this study, we present CRISPRdirect (http://crispr.dbcls.jp/),which provides efficient selection of CRISPR/Cas target sites with
reduced numbers of potential off-target candidates. CRISPRdirect
investigates the entire genome for perfect matches with each candidate target sequence (20 mer) and their seed sequence (12 or 8 mer)flanking the PAM. Users can also browse the detailed list of potential off-target sites that have partial complementarity with theselected sequence. The server incorporates genomic sequences ofhuman, mouse, rat, marmoset, pig, chicken, frog, zebrafish, Ciona,fruit fly, silkworm, Caenorhabditis elegans, Arabidopsis, rice,

C The Author 2014. Published by Oxford University Press.
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This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits
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Fig. 1. Screenshot from the CRISPRdirect web server. (A) Top page. The server accepts either an accession number or a nucleotide sequence as input. (B) Typical
output of CRISPRdirect. A list of CRISPR/Cas target candidates is displayed. (C) A graphical view of target sites demonstrates the position and orientation of eachsite. (D) The results can be exported as tab-delimited text or in JSON format. (E) Detailed list of potential off-target sites which visualizes the positions of mismatches and gaps

Sorghum and budding yeast. Currently, several web servers areavailable for designing CRISPR/Cas gRNAs (Supplementary Table
S1). CRISPR Design (Hsu et al., 2013; Ran et al., 2013) performs
gRNA selection from an input sequence up to 250 bp, and gRNAs
are scored based on predicted off-target interactions. E-CRISP(Heigwer et al., 2014) ranks gRNAs according to on-target specificity and number of off-targets. E-CRISP, ZiFiT (Sander et al.,2010), Cas9 Design (Ma et al., 2013) and CHOPCHOP (Montague
et al., 2014) utilize Bowtie (Langmead and Salzberg, 2012) to perform off-target searches allowing mismatches. On the other hand,DNA2.0 gRNA Design Tool (https://www.dna20.com/eCommerce/startCas9) searches for perfect matches with 12 nt seed to identify
off-target sites. These servers except CRISPR Design and ZiFiT canprocess at least 10 kbp of input sequence. Web servers for checking
off-target sites for given 20 nt sequences are also available, suchas Cas-OFFinder (Bae et al., 2014) and GGGenome (http://
GGGenome.dbcls.jp/). These web servers are useful for designing
gRNAs for a few input sequences, but processing large number of input sequences requires a laborious process. Even in such cases,
CRISPRdirect returns the results quickly and provides a convenientinterface for automated gRNA design as described in the Data export and API section, making it a powerful tool for using CRISPR/
Cas system on a genome-wide scale.

2 Web Server Implementation
2.1 Overview
The web server accepts an accession number, a genome coordinate
or an arbitrary nucleotide sequence up to 10 kbp as input (Fig. 1A)
and returns a list of CRISPR/Cas target candidates. Target sequencesof 20 nt adjacent to the PAM sequence (e.g. NGG, NRG) aresearched from both strands of the input sequence and listed asshown in Figure 1B. The list contains target position, target sequence, additional information on the sequence and the number oftarget sites in the genome. The additional information on the sequences such as GC content and calculated melting temperature(Tm) are provided, since previous report suggested that sgRNA sequences with very high or low GC content were less effective against
their targets (Wang et al., 2014). The presence or absence of TTTT

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B

Software for designing CRISPR/Cas guide RNA(four consecutive T’s that cause pol III termination) in the target sequence is also indicated in order to avoid TTTT in gRNA vectors
with pol III promoter. A detailed description of the web server isprovided in Supplementary Methods.

2.2 Off-target evaluation

off-target sites (Fig. 1E) and select the sequence that has fewer
off-target hits on unrelated loci. Alternatively, if site-specific gRNA
could not be designed within intended region, multiple gRNA
approaches would be considerable (Guilinger et al., 2014; Ranet al., 2013; Tsai et al., 2014). For such strategy, graphical view of
CRISPRdirect results which visualizes the position and orientationof target sites (Fig. 1C) would be helpful for selecting paired
gRNAs.

2.3 Data export and API
The results can be exported as tab-delimited text or in JSON format
from the bottom of the result page (Fig. 1D). Users can copy–paste
the text results into a spreadsheet or text editor for downstream
analysis. The results can also be downloaded as a separate file byclicking the ‘download’ link. Alternatively, tab-delimited text or
JSON output can be obtained via API, which is convenient forusers to design a number of CRISPR/Cas targets in an automatedmanner.

Funding
Life Science Database Integration Project, National Bioscience Database
Center (NBDC) of Japan Science and Technology Agency (JST) (to Y.N. andH.B.); Grant-in-Aid for Scientific Research from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT) of Japan (to Y.N. andK.U.-T.); Cell Innovation Program of MEXT (to K.U.-T.).
Conflict of interest: none declared.

References
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The number of target sites in the genome (Fig. 1B) is counted
using Jellyfish (Marçais and Kingsford, 2011). The column ‘20merþPAM’ shows the number of hits with perfect matches foreach target sequence (20 mer) adjacent to the PAM. Although theexact length of the completely complementary region necessary
for cleavage by CRISPR nucleases is unknown, the mutations
within the ‘seed’ sequence at 8–12 nt immediately adjacent to the
PAM are known to impair cleavage, suggesting that this region is
the most critical determinant of target specificity (Cong et al.,2013; Fu et al., 2013; Hsu et al., 2013; Pattanayak et al., 2013).
Therefore, we built up the columns ‘12 merþPAM’ and ‘8merþPAM’ in order to show the number of hits with perfectmatches for their seed sequence (12 or 8 mer, respectively) adjacent to the PAM. Note that the numbers of hits displayed here include both on-target and off-target sites. For instance, one (‘1’) in
these columns indicates that the sequence has only one perfectmatch with the intended target site. Any number greater than one
indicates that there are some potential off-target sites. Thus, interms of avoiding off-target editing, the smaller the number (butnot zero) is, the better. Zero (‘0’) in these columns means that thesequence has no match in the genomic sequence; such sequences
may possibly span over exon–exon junctions, so their use should
be avoided. CRISPRdirect highlights the CRISPR/Cas targets thathave relatively fewer off-target sites (Fig. 1B and C). A detailed
list of off-target candidates can be investigated by clicking the‘detail’ link (Fig. 1E). The searches allowing mismatches and gaps
(insertions and/or deletions) are performed using GGGenome(http://GGGenome.dbcls.jp/) REST API developed by the authors’group instead of widely used BLAST (Altschul et al., 1990), because BLAST may overlook some potential off-targets as mentioned in our previous work describing siDirect (Naito et al.,2004), a web server for designing functional siRNA with reduced
off-target effects. GGGenome quickly searches short nucleotide
sequences utilizing suffix arrays and inverse suffix links indexedon solid state drive (SSD). As shown in Supplementary Table S1,
off-target searches allowing gaps are not yet available in otherexisting web tools. However, the most recent report shows that
CRISPR/Cas9 system has off-target activity with insertions or deletions between target DNA and gRNA sequences (Lin et al.,2014). Therefore, we consider that off-target searches allowing
mismatches and gaps would be a more suitable procedure to list
off-target candidates exhaustively. The positions of the mismatches and gaps are visualized in the list (Fig. 1E), which may
help predict the potency of off-target editing.
CRISPRdirect incorporates genomic sequences of various organisms to perform off-target searches. Although Xenopus laevis haslong been used as a preferred model organism among developmental
biologists, we incorporated X.tropicalis genome instead of X.laevis
genome, because X.tropicalis is diploid while X.laevis is allotetraploid which makes it difficult to select specific targets.
There are some loci that are difficult to select specific targets.
Typical examples are the histone clusters (NM_021059, etc.) andribosomal proteins (NM_022551, etc.), which are known to form
multigene families. When designing CRISPR targets for such genes,
users should manually investigate a detailed list of potential

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