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
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