A detailed cell-free transcription-translation-based assay to decipher CRISPR protospacer-adjacent motifs.
Cast your vote
You can rate an item by clicking the amount of stars they wish to award to this item.
When enough users have cast their vote on this item, the average rating will also be shown.
Your vote was cast
Thank you for your feedback
Thank you for your feedback
MetadataShow full item record
AbstractThe RNA-guided nucleases derived from the CRISPR-Cas systems in bacteria and archaea have found numerous applications in biotechnology, including genome editing, imaging, and gene regulation. However, the discovery of novel Cas nucleases has outpaced their characterization and subsequent exploitation. A key step in characterizing Cas nucleases is determining which protospacer-adjacent motif (PAM) sequences they recognize. Here, we report advances to an in vitro method based on an E. coli cell-free transcription-translation system (TXTL) to rapidly elucidate PAMs recognized by Cas nucleases. The method obviates the need for cloning Cas nucleases or gRNAs, does not require the purification of protein or RNA, and can be performed in less than a day. To advance our previously published method, we incorporated an internal GFP cleavage control to assess the extent of library cleavage as well as Sanger sequencing of the cleaved library to assess PAM depletion prior to next-generation sequencing. We also detail the methods needed to construct all relevant DNA constructs, and how to troubleshoot the assay. We finally demonstrate the technique by determining PAM sequences recognized by the Neisseria meningitidis Cas9, revealing subtle sequence requirements of this highly specific PAM. The overall method offers a rapid means to identify PAMs recognized by diverse CRISPR nucleases, with the potential to greatly accelerate our ability to characterize and harness novel CRISPR nucleases across their many uses.
CitationMethods. 2018;143:48-57. doi:10.1016/j.ymeth.2018.02.016.
AffiliationHIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
JournalMethods (San Diego, Calif.)
The following license files are associated with this item:
- Creative Commons
Except where otherwise noted, this item's license is described as Attribution-NonCommercial-ShareAlike 4.0 International
- Rapid and Scalable Characterization of CRISPR Technologies Using an E. coli Cell-Free Transcription-Translation System.
- Authors: Marshall R, Maxwell CS, Collins SP, Jacobsen T, Luo ML, Begemann MB, Gray BN, January E, Singer A, He Y, Beisel CL, Noireaux V
- Issue date: 2018 Jan 4
- Scalable characterization of the PAM requirements of CRISPR-Cas enzymes using HT-PAMDA.
- Authors: Walton RT, Hsu JY, Joung JK, Kleinstiver BP
- Issue date: 2021 Mar
- Developing Heritable Mutations in Arabidopsis thaliana Using a Modified CRISPR/Cas9 Toolkit Comprising PAM-Altered Cas9 Variants and gRNAs.
- Authors: Yamamoto A, Ishida T, Yoshimura M, Kimura Y, Sawa S
- Issue date: 2019 Oct 1
- A pipeline for characterization of novel Cas9 orthologs.
- Authors: Karvelis T, Young JK, Siksnys V
- Issue date: 2019
- A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing.
- Authors: Edraki A, Mir A, Ibraheim R, Gainetdinov I, Yoon Y, Song CQ, Cao Y, Gallant J, Xue W, Rivera-Pérez JA, Sontheimer EJ
- Issue date: 2019 Feb 21