FORCAST: a fully integrated and open source pipeline to design Cas-mediated mutagenesis experiments

Abstract

Cas-mediated genome editing has enabled researchers to perform mutagenesis experiments with relative ease. Effective genome editing requires tools for guide RNA selection, off-target prediction, and genotyping assay design. While independent tools exist for these functions, there is still a need for a comprehensive platform to design, view, evaluate, store, and catalogue guides and their associated primers. The Finding Optimizing and Reporting Cas Targets (FORCAST) application integrates existing open source tools such as JBrowse, Primer3, BLAST, bwa, and Silica to create a complete allele design and quality assurance pipeline. FORCAST is a fully integrated software that allows researchers performing Cas-mediated genome editing to generate, visualize, store, and share information related to guides and their associated experimental parameters. It is available from a public GitHub repository and as a Docker image, for ease of installation and portability.

Impact of Essential Genes on the Success of Genome Editing Experiments Generating 3,313 New Genetically Engineered Mouse Lines

“…Guide RNA (gRNA) spacer sequences were selected using either the CRISPR design tool 30 , the Wellcome Sanger Genome Editing (WGE) Tool 31 , CRISPOR 32 , CRISPRTools 33 , CHOPCHOP 34 , or FORCAST 35 . Suitable gRNA spacer sequences were selected to minimize predicted off-target mutagenesis using specificity scores >65 when available and/or sequences with at least 3 mismatches for all predicted off-target sites.…”