Work

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

Year: 2020

Type: article

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).

Source: STAR Protocols

DOI: 10.1016/j.xpro.2019.100003

Authors: Ryan Marshall, Chase L. Beisel, Vincent Noireaux

Institutions: University of Minnesota, University of Würzburg, Helmholtz Institute for RNA-based Infection Research

Language: English

Topic: CRISPR and Genetic Engineering

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $1,550

Funders: Defense Advanced Research Projects Agency, Advanced Research Projects Agency