University of Minnesota
School of Physics & Astronomy

Physics and Astronomy Calendar

Thursday, January 25th 2018
10:10 am:
Biophysics Seminar in 120 PAN
Speaker: Ryan Marshall, UMN
Subject: Rapid and scalable characterization of CRISPR technologies using a cell-free transcription-translation system (TXTL)

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. 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 use 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 diverse anti-CRISPR proteins, develop a fast and scalable screen for protospacer-adjacent motifs, and show that dCas9 bound to a protospacer can be displaced. These examples underscore how TXTL can facilitate the characterization and application of CRISPR technologies across their many uses.

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