An Enhanced Assay to Characterize Anti-CRISPR Proteins Using a Cell-Free Transcription-Translation System

CRISPR-Cas systems were originally described as a part of the immune system of bacteria, but with time gave rise to one of the most important biotechnological genome-editing tools to-date. The characterization of CRISPR-Cas immune systems in bacteria was quickly followed by the discovery of anti-CRISPR proteins (Acrs) in bacteriophages. These proteins block different steps of CRISPR-based immunity and, as some inhibit Cas nucleases, can offer tight control over CRISPR technologies. Currently, only a handful of Acrs have been discovered against a small fraction of CRISPR-Cas systems while many more Acrs likely exist against the multitude of Cas nucleases in nature. Unfortunately, current methods to identify putative Acrs often include time-consuming steps like protein purification or in vivo experiments. Here, we report a rapid and scalable method for characterizing putative Acrs against Cas nucleases using an E. coli-derived cell-free transcription-translation system. Using known Acrs against Cas9 as models, we demonstrate how the method can be used to measure the inhibitory activity of individual Acrs in under two days. We also show how the method can overcome non-specific inhibition of gene expression observed for some Acrs. We have also been applying the method to rapidly interrogate Acr candidates against Cas12a. Our results conform with previously published data and therefore prove the efficacy of our method and pave the way for future experiments testing Acr candidates against CRISPR-Cas systems that still lack the discovery of Acrs. In total, the method should accelerate the interrogation and application of Acrs as CRISPR-Cas inhibitors and represents a distinct use of cell-free systems.