Extended Data Fig. 2: Generalization of the CAA.
From: A general approach to identify cell-permeable and synthetic anti-CRISPR small molecules
(a) Demonstration of FnCas12a CAA. The fluorescence of the FnCas12a-specific substrate labelled on the non-targeting strand (NTS) is not quenched in the presence of quencher unless the duplex is disrupted by cleavage via an active FnCas12a:gRNA complex. A single DNA strand containing the fluorophore (SS-DNA) can be completely quenched in the absence of an unlabelled complementary strand. Error bars represent mean ± SD from 4 independent replicates. For FnCas12a:gRNA (4th bar) compared to FnCas12a only (3rd bar), p = 1.5 × 10−7 (unpaired t-test, two-tailed). (b) Demonstration of FnCas12a CAA. The fluorescence of the FnCas12a-specific substrate labelled on the targeting strand (TS) is quenched poorly even in the presence of active FnCas12a:gRNA complex. Error bars represent mean ± SD from 4 independent replicates. For FnCas12a:gRNA (4th bar) compared to FnCas12a only (3rd bar), p = 3.3 × 10−5 (unpaired t-test, two-tailed). (c) Gel-monitored cleavage of FAM-labelled oligos (20 nM) by AsCas12a (100 nM), LbCas12a (100 nM), and FnCas12a (100 nM) in a PAM-dependent manner.