Rapid and Scalable Characterization of CRISPR Technologies Using an E. coli Cell-Free Transcription-Translation System

Mol Cell. 2018 Jan 4;69(1):146-157.e3. doi: 10.1016/j.molcel.2017.12.007.


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. Here, 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 used 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 24 diverse anti-CRISPR proteins, and develop a fast and scalable screen for protospacer-adjacent motifs that was successfully applied to five uncharacterized Cpf1 nucleases. These examples underscore how TXTL can facilitate the characterization and application of CRISPR technologies across their many uses.

Keywords: Cas9; Cascade; Cpf1; PAM; TXTL; prototyping; synthetic biology.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • CRISPR-Cas Systems / genetics*
  • Cell-Free System / metabolism*
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics
  • DNA, Bacterial / genetics
  • Endonucleases / metabolism
  • Escherichia coli / genetics*
  • Genetic Engineering / methods*
  • Oryza / genetics
  • Protein Biosynthesis / genetics*
  • RNA, Guide / genetics
  • Transcription, Genetic / genetics*


  • DNA, Bacterial
  • RNA, Guide
  • Endonucleases