Skip to main content
. Author manuscript; available in PMC: 2014 Dec 1.
Published in final edited form as: Nat Biotechnol. 2014 Apr 25;32(6):569–576. doi: 10.1038/nbt.2908

Figure 2. Design and optimization of RNA-guided FokI nucleases.

Figure 2

(a) Schematic illustrations of a ZFN, TALEN, FokI-dCas9 fusion and dCas9-FokI fusion.

(b) Screening the EGFP disruption activities of FokI-dCas9 fusion with gRNA pairs targeted to half-sites in one of two orientations: PAM-in (left panel) and PAM-out (right panel). Half-sites were separated by spacer sequences of variable lengths ranging from 0 to 31 bps. EGFP disruption was quantified by flow cytometry, n = 1. Corresponding data for the dCas9-FokI fusion and the same gRNA pairs is shown in Supplementary Fig. 1. Note the background level of EGFP-negative cells that can be observed with the control cell sample. Control shown is the same experimental sample in both of these bar graphs and in those of Supplementary Fig. 1 and is presented redundantly in each of these graphs for ease of comparison.

(c) Additional assessment of FokI-dCas9-mediated EGFP disruption activities on target sites with half-sites in the PAM-out orientation and with spacer lengths ranging from 10 to 20 bp. EGFP disruption was quantified by flow cytometry. Values shown represent the mean of duplicate measurements. Raw values of the EGFP disruption experiments can be found in Supplementary Table 2.

(d) Mean EGFP disruption values of the data from (c) grouped according to spacer length. Error bars represent s.e.m.