RESUMO
The use of CRISPR-Cas bacterial adaptive immunity system components for targeted DNA changes has opened broad prospects for programmable genome editing of higher organisms. The most widely used gene editors are based on the Cas9 effectors of the type II CRISPR-Cas systems. In complex with guide RNAs, Cas9 proteins are able to directionally introduce double-stranded breaks into DNA regions that are complementary to guide RNA sequences. Despite the wide range of characterized Cas9s, the search for new Cas9 variants remains an important task, since the available Cas9 editors have several limitations. This paper presents a workflow for the search for and subsequent characterization of new Cas9 nucleases developed in our laboratory. Detailed protocols describing the bioinformatical search, cloning, and isolation of recombinant Cas9 proteins, testing for the presence of nuclease activity in vitro, and determining the PAM sequence, which is required for recognition of DNA targets, are presented. Potential difficulties that may arise, as well as ways to overcome them, are considered.
