Type II CRISPR-Cas System Nucleases: a Pipeline for Prediction and <i>in vitro</i> Characterization

Abstract

The use of CRISPR-Cas bacterial adaptive immunity systems components for targeted DNA changing has opened broad prospects for programmable genome editing of higher organisms. The most widely used gene editor-s 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-strand breaks into DNA regions complementary to guide RNA sequences. Despite the wide range of characterized Cas9s, the search for new Cas9 variants remains an actual task, since the available Cas9 editors have several limitations. This paper presents a workflow for the search 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 nuclease activity in vitro, and determining the PAM sequence required for recognition of DNA targets, are presented. Potential difficulties that may arise, as well as ways to overcome them, are considered.