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. Author manuscript; available in PMC: 2018 Jan 9.
Published in final edited form as: Cell Rep. 2017 Dec 26;21(13):3728–3739. doi: 10.1016/j.celrep.2017.11.100

Figure 2. Time Course Assay to Quantify the RNA-Independent DNA Cleavage Activity.

Figure 2

(A) Activity of FnoCas12a on pUC19 plasmid. The major product is the nicked band. The linearization of the plasmid is incomplete after an overnight incubation.

(B) Activity of FnoCas9 on pUC19 plasmid. The linearization of the plasmid is minimal compared to FnoCas12a.

(C) Activity of FnoCas12a on M13mp18 circular DNA. The DNA is degraded, and there is no accumulation of lower molecular weight species except for the smaller degradation products.

(D) Activity of SpyCas9 on M13mp18 circular DNA. The DNA is degraded during the reaction. The graphs represent the average formation of nicked DNA for ds plasmid substrate or the amount of circular DNA left for circular ssDNA substrate. The graphs (mean ± SEM) indicate activity based on quantification of DNA bands (Schneider et al., 2012), and error bars are from three replications performed using proteins from different protein preparations.

(E) Time to reach 50% activity for all activities.

The error bars are represented as SEM and were calculated using the formula SEM=SD/n, where n represents the number of replications (see Experimental Procedures). SD was calculated using the formula SD=∑(P−PAV)2(n−1). C, control with no protein; N, nicked; L, linear; SC, supercoiled; Cr, circular ssDNA; D, degradation.