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

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

Summary: 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 fluores...

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Main Authors: Ryan Marshall, Chase L. Beisel, Vincent Noireaux
Format: Article
Language:English
Published: Elsevier 2020-06-01
Series:STAR Protocols
Online Access:http://www.sciencedirect.com/science/article/pii/S2666166719300036
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spelling doaj-6d09bc34b5fa42bf9f4cab6d58e97de62020-11-25T04:07:46ZengElsevierSTAR Protocols2666-16672020-06-0111100003Rapid Testing of CRISPR Nucleases and Guide RNAs in an E. coli Cell-Free Transcription-Translation SystemRyan Marshall0Chase L. Beisel1Vincent Noireaux2School of Physics and Astronomy, University of Minnesota, 115 Union Street SE, Minneapolis, MN 55455, USA; Corresponding authorHelmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Center for Infection Research, 97080 Würzburg, Germany; Medical Faculty, University of Würzburg, Würzburg, Germany; Corresponding authorSchool of Physics and Astronomy, University of Minnesota, 115 Union Street SE, Minneapolis, MN 55455, USA; Corresponding authorSummary: 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).http://www.sciencedirect.com/science/article/pii/S2666166719300036
collection DOAJ
language English
format Article
sources DOAJ
author Ryan Marshall
Chase L. Beisel
Vincent Noireaux
spellingShingle Ryan Marshall
Chase L. Beisel
Vincent Noireaux
Rapid Testing of CRISPR Nucleases and Guide RNAs in an E. coli Cell-Free Transcription-Translation System
STAR Protocols
author_facet Ryan Marshall
Chase L. Beisel
Vincent Noireaux
author_sort Ryan Marshall
title Rapid Testing of CRISPR Nucleases and Guide RNAs in an E. coli Cell-Free Transcription-Translation System
title_short Rapid Testing of CRISPR Nucleases and Guide RNAs in an E. coli Cell-Free Transcription-Translation System
title_full Rapid Testing of CRISPR Nucleases and Guide RNAs in an E. coli Cell-Free Transcription-Translation System
title_fullStr Rapid Testing of CRISPR Nucleases and Guide RNAs in an E. coli Cell-Free Transcription-Translation System
title_full_unstemmed Rapid Testing of CRISPR Nucleases and Guide RNAs in an E. coli Cell-Free Transcription-Translation System
title_sort rapid testing of crispr nucleases and guide rnas in an e. coli cell-free transcription-translation system
publisher Elsevier
series STAR Protocols
issn 2666-1667
publishDate 2020-06-01
description Summary: 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).
url http://www.sciencedirect.com/science/article/pii/S2666166719300036
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AT vincentnoireaux rapidtestingofcrisprnucleasesandguidernasinanecolicellfreetranscriptiontranslationsystem
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