Fig. 2: Functional assessment of split sgRNA demonstrating equivalent trans-cleavage activity to full-length sgRNA.
From: Regulating cleavage activity and enabling microRNA detection with split sgRNA in Cas12b
a Denaturing PAGE analysis demonstrates the effective trans-cleavage activity of tracrDR+Spacer and tracrRNA+DR+Spacer, comparable to that of full-length sgRNA and tracrRNA+crRNA. FAM-ssDNA denotes a FAM-labeled single-stranded random DNA strand that is cleaved upon activation of the trans-cleavage activity of the Cas12b system. dsDNA target refers to the double-stranded target DNA that is targeted by the sgRNA, whereas Non-target indicates double-stranded DNA that is not recognized by sgRNA. The experiment was performed three times. b Fluorescent signal measurements provide supporting evidence for the trans-cleavage activity for both tracrDR+Spacer and tracrRNA+DR+Spacer, with results comparable to those observed for full-length sgRNA and tracrRNA+crRNA. This system includes a reporter (F-Q) whose fluorescence is released following the digestion action of Cas12b. (+) signifies the presence of the optimal double-stranded target DNA, while (−) denotes double-stranded DNA that is not targeted by the sgRNA. Data are shown as mean value +/− SD (n = 3, biologically independent samples). c Michaelis–Menten kinetic curves illustrate the relationship between Cas12b reaction velocity and substrate concentration when using either full-length or split sgRNA. All the experiments were conducted in triplicate and error bars represent mean value +/− SD (n = 3). d Comparative analysis of the Michaelis–Menten kinetic parameters for Cas12b employing either full-length or split sgRNA. Source data are provided as a Source Data file.
