1 Introduction

The bacterial adaptive immune system, CRISPR (clustered regularly interspaced short palindromic repeats), was recently developed as a powerful and multi-purpose technology for genome engineering, including editing (modifying the genomic sequence) (Cong et al., 2013; Mali et al., 2013), and regulation (repressing or activating expression of genes) (Gilbert et al., 2013, 2014; Qi et al., 2013). The system is highly programmable, utilizing a single protein, the nuclease Cas9 for editing or the nuclease-deficient dCas9 for regulation. A single guide RNA (sgRNA) is required for precise and programmable DNA targeting (Doudna and Charpentier, 2014). Effective and specific genome engineering requires careful design of sgRNAs, which remains a major challenge. Computational tools have been used to facilitate the design of sgRNAs for CRISPR editing but not for other applications such as transcriptional regulation. These computational tools should enable automated sgRNA design and off-target site validation (Bae et al., 2014; Doench et al., 2014; Heigwer et al., 2014; O'Brien and Bailey, 2014; Xiao et al., 2014). A major goal of our designer tool is to address the discrepancy for designing sgRNAs that allow efficient and highly specific repression or activation of genes and for generating genome-wide sgRNA libraries for genetic screening in different organisms.

Here, we describe CRISPR-ERA webserver, an automated and comprehensive sgRNA design tool for CRISPR-mediated editing, repression, and activation (ERA) (Fig. 1). CRISPR-ERA utilizes a fast algorithm to search for genome-wide sgRNA binding sites and evaluates their efficiency and specificity using a set of rules summarized from published data for CRISPR editing, repression and activation (Cong et al., 2013; Doudna and Charpentier, 2014; Gilbert et al., 2014; Qi et al., 2013; Ran et al., 2013). The design features are annotated and the target sites can be visualized in a genome browser. We also provide a local version for the generation of whole-genome sgRNA libraries.

2 Methods

For each target gene or genomic site, CRISPR-ERA first searches all targetable sites in that particular organism for patterns of N₂₀NGG (N = any nucleotide). Each target sequence is then calculated for two scores (Supplementary Methods): (i) an efficacy score (E-score) based on the sequence features such as GC content (%GC), presence of poly-thymidine (which is a terminator for effective transcription of sgRNAs), and location information such as the distance from target gene transcriptional start sites (TSS); and (ii) a specificity score (S-score) based on the genome-wide off-target binding sites. For each sgRNA design, we compute the genome-wide sequences that contain an adjacent NRG (R = A or G) protospacer adjacent motif (PAM) site and zero, one, two, or three mismatches complementary to the sgRNA using Bowtie (Langmead et al., 2009), which are regarded as off-target binding sites. The penalty score for NAG off-target is smaller than NGG off-target. The sgRNAs are finally ranked by the sum of E-score and S-score (Fig. 1; Supplementary Fig. S1).

We implement a user-friendly web server (http://CRISPR-ERA.stanford.edu) that hosts the web application for the sgRNA designer tool. The webserver will host a broad category of sequenced organisms. Currently, it provides sgRNA design service for nine most commonly used prokaryotic and eukaryotic organisms including Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, Danio rerio, Rattus norvegicus, Mus musculus, and Homo sapiens, etc. (Supplementary Table S1.). The web application enables rapid searching in the pre-assembled sgRNA database using an indexing searching approach (Supplementary Methods). The program outputs the sequence, target location, off-target details of the possible sgRNAs, their E- and S-scores etc. Results can be visualized using the UCSC genome browser to highlight the custom tracks (Supplementary Fig. S2 and S3).

3 Conclusions

CRISPR-ERA enables easy, fast, and predictive design of sgRNAs for broad applications of CRISPR in genome editing, gene repression and activation. The tool can be applied to other types of CRISPR applications such as genome imaging (Chen et al., 2013) and CRISPR synthetic circuit design (Kiani et al., 2014), and expanded to other organisms. We also provide the source code for the generation of whole-genome sgRNA libraries, useful for genome-wide screening based on CRISPR, CRISPRi or CRISPRa (Gilbert et al., 2014, Shalem et al., 2013; Wang et al., 2013).

Supplementary Material

Funding

L.S.Q. acknowledges support from NIH Office of The Director (OD), and National Institute of Dental & Craniofacial Research (NIDCR). H.L., Z.W., Y.L. and X.W. are supported by the 973 program (2012CB316503>), NSFC (grant number 61322310>, 31371341>) and FANEDD (grant number 201158>). This work was also supported by NIH Director’s Early Independence Award (grant number OD017887>, L.S.Q.) and NIH R01 (grant number DA036858>, L.S.Q.).

Conflict of Interest: none declared.