Although the CRISPR/Cas9 system has many advantages for use in gene editing including efficiency, simplicity and target specificity, the potential for off-target effect (via Cas9 enzyme binding to unintended genomic sites for cleavage and inducing mutations) also has a certain influence on clinical applications. Therefore, it is important to consider potential off-target effects and to verify if off-target cleavage has occurred.
To address testing for off-target cleavage, did you know that it is possible to use a DISCOVER-seq (discovery of in situ Cas off-targets and verification by sequencing) approach when using CRISPR/Cas9 technology for gene editing?
A detailed experimental protocol using the 4D-Nucleofector® Device and analysis pipeline was recently published for the use of ribonucleoproteins (RNPs), but in theory, it can be used with any type of CRISPR-Cas delivery vehicle (e.g. lipofection, viral transduction). Furthermore, any form of editing reagent (e.g. RNPs, plasmids, mRNAs) can be used with the DISCOVER-Seq workflow.
This method is based on tracking the precise recruitment of MRE11 to double-strand breaks (DSBs) by chromatin immunoprecipitation and subsequent next-generation sequencing. BLENDER (blunt end finder) as a customized open-source bioinformatics pipeline is then able to identify off-target sequences genome-wide in primary cells and in situ. There are three advantages of this testing approach including low false-positive rates, the application of the method to a wide range of systems like cells from patients and animal models, and the rapidity of the test method in that the whole protocol can be completed within 2 weeks.
Please keep in mind, it is quite important to consider the off-target effect and to select your sgRNAs wisely with a low off-target effect and high on target effect.
If you need help with your CRISPR experiments, for more information concerning sgRNA design tools and troubleshooting/optimizing your transfection efficiency, contact us.
Looking forward to your results in knock-ins, outs, base editors, epigenetic editing, screenings, etc. by the use of the CRISPR tool like a ‘Swiss army knife’.
Scientific Support Specialist
Wienert B, Wyman SK, Yeh CD, Conklin BR, Corn JE. CRISPR off-target detection with DISCOVER-seq. Nat Protoc. 2020 May;15(5):1775-1799