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

Marshall, R., University of Minnesota
Maxwell, C., North Carolina State University
Collins, S., North Carolina State University
Luo, M. L., North Carolina State University
Jacobsen, T., North Carolina State University
Beisel, C. L., North Carolina State University
Noireaux, V., University of Minnesota
We present the use of E. coli cell-free transcription-translation systems (TXTL) to vastly improve the speed and scalability of CRISPR characterization and validation. Unlike prior approaches that require protein purification or live cells, TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of gene repression. To demonstrate the applicability of TXTL, we rapidly measure guide RNA-dependent DNA cleavage and gene repression for single- and multi-effector CRISPR-Cas systems, accurately predict the strength of gene repression in E. coli, quantify the inhibitory activity of anti-CRISPR proteins, and develop a fast and scalable high-throughput screen for protospacer-adjacent motifs. These examples underscore the potential of TXTL to facilitate the characterization and application of CRISPR technologies across their many uses.