In Vitro Cas9 Cleavage Assay to Check Guide RNA Efficiency

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) system has become a popular toolkit for editing genomes of interest in a wide variety of organisms. The Cas9 endonuclease enzyme is targeted to a specific genomic region by a small single guide RNA (sgRNA). The cleavage efficiency of Cas9 varies greatly from one sgRNA to another sgRNA. Mutagenesis rate of a CRISPR-Cas experiment strongly depends on the sgRNA used. Presently accessible web-based tools for sgRNA design predict a wide variety of candidate sgRNAs for a single genomic target site. Despite these in silico predictions, not every sgRNA displays the same cleavage efficiency. To encounter this discrepancy, here, we present an in vitro method to screen multiple sgRNAs to identify the most suitable one that can efficiently introduce a double-stranded break at a particular genomic target site. This screening method allows a researcher to choose the best one among several online predicted sgRNAs prior to deliver genome editing reagents into live plant or animal cells.