Extended Data Fig. 7: Additional data related to the characterization of real-time dynamics of mRNA localization by CRISPR-TO in Fig. 2j,k.
From: Programmable control of spatial transcriptome in live cells and neurons
a, Box-and-whisker plots depicting the percentage of dCas13 protein (green) and endogenous GAPDH mRNA (red) localized on the OMM in individual cells. HeLa cells were transfected with dCas13, MAVS*-PYL1, and gG123 with different ABA concentrations for 4 h. Each dot represents one cell. n, cell number. Two-sided, unpaired Student’s t-test between 0 µM and indicated groups. The curves are fitted with a saturation binding curve with Hill coefficient, with assumption that the background values are the mean of the 0 µM group. Box-and-whisker plots: median, 25% to 75% boxes, whiskers (10–90%), and outliers indicated. b, Box-and-whisker plots depicting the percentage of dCas13 protein localized on the OMM over time. HeLa cells expressing dCas13, MAVS*-PYL1, and gG123 were treated with 250 µM ABA for indicated durations. Localization dynamics fitted by a two-phase exponential model; second-phase parameters (characteristic time τ, background A0, and maximum activation amplitude Am) constrained by mRNA localization data (Fig. 2j, left). n, cell number. Two-sided, unpaired Student’s t-test between 0 h and indicated groups. c, Time-course plot showing percentage of OMM-localized free (orange) and mRNA-bound (blue) dCas13 proteins over time after ABA treatment. Curves were generated by a simulation based on the two-phase model (Extended Data Fig. 7b and Supplementary Methods). d, Box-and-whisker plots depicting the percentage of dCas13 protein localized on the OMM over time. HeLa cells expressing dCas13, MAVS*-PYL1, and gG123 were treated with 250 µM ABA for 4 h followed by ABA removal for indicated durations. Rightmost group represents cells treated with DMSO for 4 h. Dissociation curve fitted by simple exponential decay model with baseline constrained to DMSO mean. n, cell number. Two-sided, unpaired Student’s t-test between 0 h and indicated groups. For a,b,d, only cells co-expressing dCas13 and MAVS*-PYL1 were analyzed. e, Schematic illustrating the MS2-tagged reporter mRNA used for real-time tracking of CRISPR-TO-mediated mRNA localization in live cells. f, Representative microscopic images showing MS2 reporter mRNA accumulation at the minus ends of microtubules (centrosome, arrows) after ABA addition. Scale bar, 20 µm. Insets (dashed outlines) show magnified views of the centrosome region (arrow). g, Quantification of the fluorescence intensity and the fraction of MS2 reporter mRNA located at the minus ends of microtubules in Extended Data Fig. 7f over time after adding ABA. h, Representative trajectories of 60 MS2 reporter mRNA particles detected in the cell shown in Extended Data Fig. 7f. The trajectories are exhibited with their traversed distance (see Supplementary Methods for calculating traversed distance) from smallest to largest. The first 20 trajectories were categorized as type I which shows confined, sub-diffusive motion of mRNA particles due to crowding cytoplasmic environment. The last 20 trajectories were categorized as type II which shows super-diffusive motion of mRNA particles corresponding to the active transport along microtubules via motor proteins. The middle 20 trajectories were likely a mixture of the two types. i, Mean squared displacement (MSD) analysis of 20 representative trajectories for two identified MS2 reporter mRNA particle populations (type I: confined/sub-diffusive; type II: active transport/super-diffusive). Lines represent linear fits of the first three MSD points. n = 20 trajectories for type I and II, respectively. Data are presented as mean values ± standard deviation. j, Distance of five representative type I and type II MS2 reporter mRNA trajectories to the centrosome (minus end of microtubules) over time.
