Extended Data Fig. 1: Validation of the CAA.
From: A general approach to identify cell-permeable and synthetic anti-CRISPR small molecules
(a) Schematic of the differently labelled PAM fluorescence polarization substrates. (b) Fluorescence polarization assay comparing 0-PAM and 12-PAM substrates with SaCas9 at multiple concentrations of unlabelled ligand. Substrates were labelled with FAM on the 3′ end. SaCas9 showed specificity for the 12-PAM substrate that decreased with increasing amounts of unlabelled competitor. Error bars represent mean ± SD from 3 independent replicates. For ‘0x UL’ compared to ‘No SaCas9’, p = 0.061 with the 0-PAM DNA and p = 1.1 × 10−4 with the 12-PAM DNA (unpaired t-test, two-tailed). (c) Fluorescence polarization assay comparing 0-PAM and 12-PAM substrates with FnCas12a at multiple concentrations of unlabelled ligand. Substrates were labelled with FAM on the 3′ end. FnCas12a showed no specificity dependent upon the presence of PAM-binding sites. Error bars represent mean ± SD from 3 independent replicates. For ‘0x UL’ compared to ‘No FnCas12a’, p = 3.5 × 10−5 with the 0-PAM and p = 7.4 × 10−7 with the 12-PAM DNA (unpaired t-test, two-tailed). (d) Fluorescence polarization assay comparing 0-PAM and 12-PAM substrates with FnCas12a at multiple concentrations of unlabelled ligand. Substrates were labelled with FAM on the 5′ end. FnCas12a showed no specificity dependent upon the presence of PAM-binding sites. Error bars represent mean ± SD from 3 independent replicates. For ‘0x UL’ compared to ‘No FnCas12a’, p = 6.2 × 10−4 with the 0-PAM and p = 6.2 × 10−5 with the 12-PAM DNA (unpaired t-test, two-tailed). (e) Inhibition of SpCas9 by AcrIIA11 monitored by the CAA. Error bars represent mean ± SD from 4 independent replicates. For 10 µM AcrIIA11 compared to buffer only, p = 1.6 × 10−7 (unpaired t-test, two-tailed). (f) The fluorescence of the SaCas9-specific substrate is not quenched in the presence of quencher unless the duplex is disrupted by cleavage via an active SaCas9: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 SaCas9:gRNA (4th bar) compared to SaCas9 only (3rd bar). p = 1.1 × 10−8 (unpaired t-test, two-tailed).
