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. Marshall et al. demonstrate that an E. coli cell-free transcription-translation (TXTL) system can be used to improve the speed and scalability of characterizing CRISPR nucleases and their accessory factors. The method will facilitate the discovery of uncharacterized CRISPR nucleases and anti-CRISPR proteins and aid the validation of designed gRNAs.
Bibliographical noteFunding Information:
We thank Ryan Leenay for help generating the FnCpf1 PAM wheel, Jennie Fagan for cloning the plasmid expressing SpyCas9, and Luciano Maraffini for providing the pCas9 plasmid (Addgene plasmid #42876). Preliminary experiments were performed during the 2016 CSHL Synthetic Biology summer course. This material is based upon work supported by the Defense Advanced Research Projects Agency (contract HR0011-16-C-01-34 , V.N. and C.L.B.), the Office of Naval Research (award N00014-13-1-0074 , V.N.), the NIH (grant 1R35GM119561-01 to C.L.B.), and the National Science Foundation (grant CBET-1403135 to C.L.B.).
- synthetic biology