Rapid cell-free characterization of multi-subunit CRISPR effectors and transposons

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 disc...

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Bibliographic Details
Published in:Molecular cell 2022-02, Vol.82 (6)
Main Authors: Wimmer, Franziska, Mougiakos, Ioannis, Englert, Frank, Beisel, Chase L.
Format: Article
Language:English
Subjects:
BASIC BIOLOGICAL SCIENCES
Cascade
CAST
high-throughput screen
PAM
PAM wheel
PAM-DETECT
restriction enzyme
TXTL
Type I CRISPR-Cas system
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Summary: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, in this work, 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.
ISSN:1097-2765