Rapid and Scalable Characterization of CRISPR Technologies Using an E. coli Cell-Free Transcription-Translation System

CRISPR-Cas systems offer versatile technologies for genome engineering, yet their implementation has been outpaced by ongoing discoveries of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation (TXTL) systems to vastly improve the speed and scalability of CRISPR characterization and validation. TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression—all without protein purification or live cells. We used TXTL to measure the dynamics of DNA cleavage and gene repression for single- and multi-effector CRISPR nucleases, predict gene repression strength in E. coli, determine the specificities of 24 diverse anti-CRISPR proteins, and develop a fast and scalable screen for protospacer-adjacent motifs that was successfully applied to five uncharacterized Cpf1 nucleases. These examples underscore how TXTL can facilitate the characterization and application of CRISPR technologies across their many uses. [Display omitted] •Active expression of multiple CRISPR nucleases and gRNAs in an E. coli TXTL system•Repression activity of dCas9 strongly correlated between TXTL and E. coli•A TXTL-based PAM assay allowed characterization of five Cpf1 nucleases•Determined specificities of 24 anti-CRISPR proteins against five Cas9 nucleases Marshall et al. demonstrate that an E. coli cell-free transcription-translation (TXTL) system can be used to improve the speed and scalability of characterizing CRISPR nucleases and their accessory factors. The method will facilitate the discovery of uncharacterized CRISPR nucleases and anti-CRISPR proteins and aid the validation of designed gRNAs.