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Rapid Testing of CRISPR Nucleases and Guide RNAs in an Cell-Free Transcription-Translation System.
Marshall, Ryan Beisel, Chase L Noireaux, Vincent
Marshall, Ryan
Beisel, Chase L
Noireaux, Vincent
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2020-06-03
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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).
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STAR Protoc. 2020 Jun 3;1(1):100003. doi: 10.1016/j.xpro.2019.100003.
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Article
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en
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2666-1667
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Attribution-NonCommercial-ShareAlike 4.0 International