gRNA-Seqret: Genome Wide Guide RNA Design and Sequence Extraction Tool

The U.S. Department of Energy (DOE) Joint Genome Institute (JGI) provides DNA sequencing and synthesis services to scientific users via community science programs. The DNA synthesis program covers all aspects of the synthetic biology design-build-test cycle, enabling users to engineer biological systems of relevance to their research and the DOE mission.

One particular product type offered by the DNA synthesis program is the design and construction of libraries with high degrees of variants. Applications of such libraries include CRISPR technologies using guide RNA (gRNA) libraries or studying structural variations of expressed proteins or regulatory elements (e.g., promoters).

Presently, there are no simple tools for extracting sequences from specific genomic regions in bulk, and most available tools for gRNA design restrict the user to pre-processed genomes from specific model organisms. To overcome these shortcomings, we have developed a web-based open-source in silico design tool: The guide RNA and Sequence Region Extraction Tool (gRNA-SeqRET). This tool allows the user to specify exactly which region of a gene, a subset of genes, or every gene in the genome for sequence extraction; a number of criteria can be used to specify gRNA targets.

The initial release of the software allows the extraction of specific sequences upstream and downstream of gene start sites and whole genome gRNA libraries for both inhibiting (“CRISPRi”) and activating (“CRISPRa”) genes in any prokaryotic organism. Furthermore, it evaluates each gRNA based on predicted RNA folding with and without the scaffold sequence, and automatically selects the top candidates. Further development in progress will extend the tools’ capabilities to use a wider variety of genome sequence and annotation file formats, the capability to process eukaryotic genomes, and the addition of more flexible sequence extraction and targeting options decoupled from the gRNA design workflow, providing a universal sequence extraction tool.