在无细胞转录-翻译系统中快速检测 CRISPR 核酸酶和向导 RNA。
Rapid Testing of CRISPR Nucleases and Guide RNAs in an Cell-Free Transcription-Translation System.
机构信息
School of Physics and Astronomy, University of Minnesota, 115 Union Street SE, Minneapolis, MN 55455, USA.
Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Center for Infection Research, 97080 Würzburg, Germany.
出版信息
STAR Protoc. 2020 Jun 3;1(1):100003. doi: 10.1016/j.xpro.2019.100003. eCollection 2020 Jun 19.
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 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).
我们提出了一个使用无细胞转录-翻译（TXTL）快速测试 CRISPR 核酸酶的 DNA 结合和切割活性的方案。通过向 TXTL 反应中添加编码核酸酶、向导 RNA 和靶向报告基因的 DNA，并在数小时内测量荧光，来评估核酸酶的活性。该反应在几微升的体积中进行，允许同时测试许多核酸酶和向导 RNA。该方案包括靶向 GFP 报告基因的(d)Cas9 的代表性结果。有关此方案的生成和使用的完整信息，请参阅 Marshall 等人的论文（2018 年）。
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