Figure 5. RNA binding and limited proteolysis of SpyCas9^WT and SpyCas9^2Pro.

A) Graph showing quantification of binary complex formed by SpyCas9^2Pro and SpyCas9^WT. EMSA was conducted using 5’−³²P labelled sgRNA^del. The protein concentration was increased from 10 nM to 70 nM relatively to sgRNA concentration (~50 nM). Graph shows the average of bound complex from three independent replications over different protein concentrations. The data indicate that the RNA binding property of SpyCas9^2Pro is not significantly reduced compared to SpyCas9^WT. B) Trypsin digestion of SpyCas9^WT and SpyCas9^2Pro with or without sgRNA. In the apo-form, the digestion profiles for both proteins are similar except for increased intensity of Band A in SpyCas9^2Pro. The sgRNA bound form of SpyCas9^2Pro is not protected to the same extent as SpyCas9^WT-sgRNA complex (see the difference in intensity of Band B). In addition, Band C is more prominent in SpyCas9^2Pro-sgRNA complex, indicating conformational differences between the two binary complexes.