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. Author manuscript; available in PMC: 2020 Jan 10.
Published in final edited form as: Cell. 2019 Jan 10;176(1-2):254–267.e16. doi: 10.1016/j.cell.2018.11.052

Figure 2. Linker length can be utilized to control Cas9-CP activity.

Figure 2.

(A) Endpoint analysis of an E. coli CRISPRi based GFP repression assay run in triplicate using Cas9- CPs identified as functional with 20 AA linkers, evaluated with GGSn linkers of length 5, 10, 15, 20, 25 and 30 AA. Error bars indicate standard deviation in all panels.

(B) Schematic describing the rationale behind using a Cas9-CP with a short AA linker as a ‘caged’ molecule.

(C) Endpoint analysis of an E. coli CRISPRi-based GFP expression time course with all six Cas9-CPs containing a 7 AA TEV linker (ENLYFQ/S) in the presence of a functional TEV protease (TEV, blue) compared with deactivated TEV protease with the catalytic triad mutant C151A (dTEV, gray) (n = 3, error bars are s.d., * = p<0.05, ns = not significant, t-test).

(D) Schematic and western blot against the Flag epitope on the C terminus of the CP-TEVs after the endpoint measurement (Figure 2C). Expected kDa indicates the predicted band size if cleavages occurs at the TEV site in the CP linker region.