Breaking-Cas—interactive design of guide RNAs for CRISPR-Cas experiments for ENSEMBL genomes

TLDR

CRISPR/Cas technology enables precise genome editing, but effective experiments depend on carefully designed guide RNAs that meet nuclease requirements and avoid off‑target binding. This study introduces Breaking‑Cas, a system for designing guide RNAs for CRISPR/Cas experiments, including Cas9 and newer nucleases. Breaking‑Cas offers unique features such as support for all ~700 ENSEMBL eukaryotic genomes, flexible PAM placement at either 5′ or 3′ ends, adjustable guide length, and nucleotide‑level scoring. The Breaking‑Cas web server is freely accessible at http://bioinfogp.cnb.csic.es/tools/breakingcas, with source code available upon request.

Abstract

The CRISPR/Cas technology is enabling targeted genome editing in multiple organisms with unprecedented accuracy and specificity by using RNA-guided nucleases. A critical point when planning a CRISPR/Cas experiment is the design of the guide RNA (gRNA), which directs the nuclease and associated machinery to the desired genomic location. This gRNA has to fulfil the requirements of the nuclease and lack homology with other genome sites that could lead to off-target effects. Here we introduce the Breaking-Cas system for the design of gRNAs for CRISPR/Cas experiments, including those based in the Cas9 nuclease as well as others recently introduced. The server has unique features not available in other tools, including the possibility of using all eukaryotic genomes available in ENSEMBL (currently around 700), placing variable PAM sequences at 5′ or 3′ and setting the guide RNA length and the scores per nucleotides. It can be freely accessed at: http://bioinfogp.cnb.csic.es/tools/breakingcas, and the code is available upon request.