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

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.

CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off‐Target Evaluation, and Strategies to Mitigate Off‐Target Effects

“…It also offers a valuable service for the assessing off‐targets of the gRNAs with the length of 18–25 nucleotides. Notably, the input form (providing a FASTA file) is very efficient and flexible (supporting the process of many sequences in a single run with multiple entries) , which is freely accessible on‐line like other tools …”

“…Notably, the input form (providing a FASTA file) is very efficient and flexible (supporting the process of many sequences in a single run with multiple entries) , which is freely accessible on-line like other tools. [93] 7.4. CasFinder…”

A history of genome editing in Saccharomyces cerevisiae

“…Fortunately, such software already exists for public use in various forms. Many biotech companies provide webtools that design gRNAs for users (ATUM, GenScript, ThermoFisher Scientific, among others), and academic researchers have produced their own gRNA evaluation tools such as ChopChop (Labun, Montague, Gagnon, Thyme, & Valen, 2016;Montague, Cruz, Gagnon, Church, & Valen, 2014), E-CRISP (Heigwer, Kerr, & Boutros, 2014) and Breaking-Cas (Oliveros et al, 2016).…”

CRISPR/Cas9: a breakthrough in generating mouse models for endocrinologists

“…A growing number of free bioinformatics tools have been developed to help in sgRNA design. The aim is to maximize the cutting efficiency while minimizing off-target cleavages (Ren et al 2014, Koo et al 2015, MacPherson & Scherf 2015, Oliveros et al 2016. The sequence of predesigned sgRNA is also available for the whole mouse exome (http://www.…”