Flexible guide-RNA design for CRISPR applications using Protospacer Workbench

805 be marked for further analysis, plotting, saving and annotation. The workbench also supports connection to the Broad Institute’s Integrative Genomics Viewer (IGV)6, which allows targets to be viewed in the context of arbitrary genome browser tracks such as genomic annotations, amino acid sequences and high-throughput sequencing data. We provide online video tutorials and written documentation to familiarize users with the Protospacer interface. The software, tutorials, mailing list and databases for several organisms are located at http:// www.protospacer.com/. The Protospacer To the Editor: The CRISPR-Cas9 endonuclease system permits genome editing at nucleotide resolution, but ensuring high efficiency while minimizing off-target cleavage requires careful design of the single guide RNA (sgRNA). Currently most researchers use online tools (e.g., E-CRISP1, CRISPR2, ZiFiT3) for sgRNA design. However, these tools have several drawbacks: the number of target-sgRNA mismatches allowed is limited; they are unable to detect every potential off-target site; they are generally limited in flexibility and throughput; and they are available for only a few organisms. Offline solutions such as sgRNAcas9 (ref. 4) provide greater flexibility and speed but require knowledge of programming and/ or command-line interfaces. Neither online nor offline software facilitates sharing of sgRNA libraries, their putative off-target sites or the statistical reports that lead to successful designs. Protospacer Workbench, an offline software for rapid, flexible design of Cas9 sgRNA, addresses all of these issues (Table 1). It combines a graphical user interface with a file-based database and third-party sequence mapping tools to maximize flexibility and information retrieval when designing sgRNAs (Fig. 1). Important design statistics are calculated, such as the off-target and Cas9-activity scores developed by Hsu et al.2 and Doench et al.5, respectively. Protospacer allows researchers to build, analyze and share their own databases of CRISPR targets, facilitating the development of custom sgRNA libraries and the transfer of CRISPR technology to new organisms. Protospacer can manage multiple databases for laboratories interested in several strains or organisms. Protospacer databases can be created from any FASTA file and annotated using either the GTF or GFF format. Each database begins as a simple catalog of the sequence and annotation data and grows through use. Protospacer’s flexible user interface is structured around four main tasks: target finding, broad target filtering, selection of candidate targets, and sgRNA design and analysis. Target finding allows searching in one or more regions of interest, defined by genomic ranges, distance to a point of interest, gene identifier, feature attribute or sequence similarity. Potential sgRNA targets found within regions of interest can be filtered by nucleotide content, by the Doench-Root activity5 score or by uniqueness in the genome. One or more of the remaining targets may Flexible guide-RNA design for CRISPR applications using Protospacer Workbench