Supplementary Figure 14: Reversible recruitment of Acr–LOV hybrid 5 to a plasma-membrane-targeted dCas9–gRNA complex.
From: Engineered anti-CRISPR proteins for optogenetic control of CRISPR–Cas9
(a) Light-inducible recruitment of a LOV–Acr hybrid fused to mCherry to a plasma-membrane-targeted dCas9–mVenus. (b) Representative fluorescence images of HEK293T cells expressing LOV–Acr hybrid 5 fused to mCherry and an mVenus–dCas9 fusion targeted to the plasma membrane. Cells were irradiated with blue light pulses every 30 s using a 488-nm laser for 20 min, followed by 20 min dark recovery. mVenus and mCherry fluorescence images were recorded every 5 min. Dashed lines indicate the nucleus boundary. Yellow boxes in the Acr–LOV–mCherry images correspond to plasma membrane close-up views shown below the images. Scale bar, 20 µm. (c) Quantification of the plasma membrane to cytoplasmic mCherry fluorescence ratio over time. Data are means ± s.e.m. n = 4 cells from biologically independent samples (cell cultures).
