Evaluation of the Efficiency of Genome Editing Tools by a Frameshift Fluorescence Protein Reporter.

Abstract

In the last decade, genome editing has been the center of attention as a novel tool for mechanistic investigations and for potential clinical applications. Various genome editing tools like meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated genes (Cas), have been developed in recent years. For the optimal use as well as continued developments of these genome editing tools, the evaluation of their efficiencies and accuracies is vital. Here, we present a protocol for a reporter based on frameshift fluorescence protein which we recently developed to evaluate the efficiency and accuracy of genome editing tools. In this method, a ~20 bp target sequence containing frame-shifting is inserted after the start codon of a cerulean fluorescence protein (CFP) to inactivate its fluorescence, and only a new insertion/deletion event in the target sequence will reactivate the CFP fluorescence. To increase the traceability, an internal ribosome entry site and a red fluorescence protein, mCherryFP, are placed downstream of the reporter. The percentage of CFP-positive cells resulted from in/del mediated fluorescence restoration can be quantified by fluorescence measuring devices as the readout for genome editing frequency. As a demonstration, we present the usage for CRISPR-Cas9 technique here with flow cytometer as the readout for fluorescence changes.