Cut Site Selection by the Two Nuclease Domains of the Cas9 RNA-guided Endonuclease. Hongfan Chen, Jihoon Choi and Scott Bailey.The Journal of Biological Chemistry, 289, 13284-13294 (2014)
4. To evaluate whether other divalent cations besides Mg2+ can activate DNA cleavage by Cas9, the authors performed plasmid cleavage assays in the presence of one of the following divalent cations: Ca2+, Mn2+, Co2+, Ni2+, and Cu2+.
a. Figure A shows cleavage of a target plasmid by Cas9 with either no metal or 1 mm of the indicated metal ions. All reactions were treated with 0.5 mm EDTA prior to metal addition. Interpret the results.
b. To identify which domain was activated, we assayed the single active site mutants of Cas9 (D9A or H599A) in a reaction buffer containing Ca2+. In both panels, the positions of negatively supercoiled (nSC), linear (L), and nicked or open circle (OC) plasmid are indicated. The linear control is a digestion of the plasmid target with the restriction enzyme AgeI.
Navigation:
