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. Author manuscript; available in PMC: 2021 Jul 16.
Published in final edited form as: Mol Cell. 2020 Jun 29;79(2):221–233.e5. doi: 10.1016/j.molcel.2020.06.014

Figure 1. Energy-Dependent Release of Cas9 and dCas9 from DNA in HSS.

Figure 1.

(A) Time course of Cas9 RNPs programmed against a linear DNA substrate in a 1:2 molar ratio in buffer, HSS with ATP, or HSS with CIP and ATPγS.

(B) Time course of Cas9 RNPs programmed against a plasmid substrate in a 1:2 molar ratio in buffer, HSS with ATP, or HSS with CIP and ATPγS.

(C) Schematic of the single-turnover competition assay.

(D) ATP-dependent unloading of dCas9 off a plasmid substrate in X. laevis HSS. Presence of linearized DNA after addition of a 10-fold molar excess of Cas9 indicates removal of dCas9 while persistence of circular DNA indicates stable binding of dCas9. See also Figure S1C.