An enhanced assay to characterize anti-CRISPR proteins using a cell-free transcription-translation system

Abstract

The characterization of CRISPR-Cas immune systems in bacteria was quickly followed by the discovery of anti-CRISPR proteins (Acrs) in bacteriophages. These proteins block different steps of CRISPR-based immunity and, as some inhibit Cas nucleases, can offer tight control over CRISPR technologies. While Acrs have been identified against a few CRISPR-Cas systems, likely many more await discovery and application. Here, we report a rapid and scalable method for characterizing putative Acrs against Cas nucleases using an E. coli-derived cell-free transcription-translation system. Using known Acrs against type II Cas9 nucleases as models, we demonstrate how the method can be used to measure the inhibitory activity of individual Acrs in under two days. We also show how the method can overcome non-specific inhibition of gene expression observed for some Acrs. In total, the method should accelerate the interrogation and application of Acrs as CRISPR-Cas inhibitors.

The New Age of Cell-Free Biology

“…Elementary circuits based on the traditional set of transcription factors, including the repressors and activators LacI, TetR, and AraC (110,139), have also been validated in TXTL. One of the most recent, compelling examples is the demonstration that CRISPR technologies can be rapidly tested in TXTL, and there is high fidelity with respect to in vivo data (140,141). This new functionality has opened the door to engineering gene circuits, such as network motifs, that include CRISPR components (114).…”

Multilevel Gene Regulation Using Switchable Transcription Terminator and Toehold Switch in Escherichia coli

“…Additionally, a synthetic terminator, T500, was selected due to its very short hairpin structure and a termination efficiency of close to 98% [45]. T500 terminator has been widely used in synthetic biology applications as an ideal terminator exhibiting a significant elongation complex (EC) dissociation rate [49][50][51]. Thus, the BBa K864600 and T500 terminators, with their strong transcription termination strengths and short stem-loop structures, can represent ideal targets to be engineered as SWT.…”

CRISPR-Cas9 Genome Editing in Human Cell Lines with Donor Vector Made by Gibson Assembly

“…At the forefront of this field are exciting, emerging issues. Chief among these, is the ability of some bacterial phage to counter the CRISPR immune response by expressing anti-CRISPR proteins that prevent DNA binding and Cas3 activity [27][28][29]. Additional functional roles for CRISPR in bacteria include promoting genome evolution, bacterial virulence and DNA repair [30][31][32][33].…”