CRISPy-web: An online resource to design sgRNAs for CRISPR applications

Abstract

CRISPR/Cas9-based genome editing has been one of the major achievements of molecular biology, allowing the targeted engineering of a wide range of genomes. The system originally evolved in prokaryotes as an adaptive immune system against bacteriophage infections. It now sees widespread application in genome engineering workflows, especially using the Streptococcus pyogenes endonuclease Cas9. To utilize Cas9, so-called single guide RNAs (sgRNAs) need to be designed for each target gene. While there are many tools available to design sgRNAs for the popular model organisms, only few tools that allow designing sgRNAs for non-model organisms exist. Here, we present CRISPy-web (http://crispy.secondarymetabolites.org/), an easy to use web tool based on CRISPy to design sgRNAs for any user-provided microbial genome. CRISPy-web allows researchers to interactively select a region of their genome of interest to scan for possible sgRNAs. After checks for potential off-target matches, the resulting sgRNA sequences are displayed graphically and can be exported to text files. All steps and information are accessible from a web browser without the requirement to install and use command line scripts.

Keywords: CRISPR; Cas9; Genome editing; Genome engineering; Guide RNA; sgRNA.

Fig. 1

Example output of a CRISPy-web run to identify sgRNAs in the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor A3(2) (NCBI GenBank ID: NC_003888.3). (A) Overview of sgRNAs identified in the complete actinorhodin gene cluster. (B) Zoom view of SCO5087 (actIORF1). (C) List of sgRNAs selected for export.