Molecular Cell 82(6) , 1210-1224.e6 ( 2022)
CRISPR-Cas biology and technologies have been largely shaped to date by the characterization and use of single-effector nucleases. By contrast, multi-subunit effectors dominate natural systems, represent emerging technologies, and were recently associated with RNA-guided DNA transposition. This disconnect stems from the challenge of working with multiple protein subunits in vitro and in vivo. Here, we apply cell-free transcription-translation (TXTL) systems to radically accelerate the characterization of multi-subunit CRISPR effectors and transposons. Numerous DNA constructs can be combined in one TXTL reaction, yielding defined biomolecular readouts in hours. Using TXTL, we mined phylogenetically diverse I-E effectors, interrogated extensively self-targeting I-C and I-F systems, and elucidated targeting rules for I-B and I-F CRISPR transposons using only DNA-binding components. We further recapitulated DNA transposition in TXTL, which helped reveal a distinct branch of I-B CRISPR transposons. These capabilities will facilitate the study and exploitation of the broad yet underexplored diversity of CRISPR-Cas systems and transposons.