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

Katharina G. Wandera, Scott P. Collins, Franziska Wimmer, Ryan Marshall, Vincent Noireaux, Chase L. Beisel

Research output: Contribution to journalArticlepeer-review

13 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)42-50
Number of pages9
StatePublished - Feb 1 2020

Bibliographical note

Funding Information:
Funding for this project was provided by DARPA , United States (contract HR0011-17-2-0042 to C.L.B.) and the Deutsche Forschungsgemeinschaft, Germany ( BE 6703/1-1 to C.L.B.).

Publisher Copyright:
© 2019 Elsevier Inc.


  • Anti-CRISPR proteins
  • Cas9
  • Genome editing
  • TXTL
  • sgRNA

PubMed: MeSH publication types

  • Journal Article
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.


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