Rapid Testing of CRISPR Nucleases and Guide RNAs in an E. coli Cell-Free Transcription-Translation System

Ryan Marshall, Chase L. Beisel, Vincent Noireaux

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

We present a protocol to rapidly test DNA binding and cleavage activity by CRISPR nucleases using cell-free transcription-translation (TXTL). Nuclease activity is assessed by adding DNA encoding a nuclease, a guide RNA, and a targeted reporter to a TXTL reaction and by measuring the fluorescence for several h. The reactions, performed in a few microliters, allow for parallel testing of many nucleases and guide RNAs. The protocol includes representative results for (d)Cas9 from Streptococcus pyogenes targeting a GFP reporter gene. For complete information on the generation and use of this protocol, please refer to the paper by Marshall et al. (2018).

Original languageEnglish (US)
Article number100003
JournalSTAR Protocols
Volume1
Issue number1
DOIs
StatePublished - Jun 19 2020

Bibliographical note

Funding Information:
The authors acknowledge the Defense Advanced Research Projects Agency , contract HR0011-16-C-01-34 (to C.L.B. and V.N.).

Publisher Copyright:
© 2019 The Author(s)

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