Fig. 1: Comprehensive assessment of PAM-mediated collateral activity of LbCas12a at 37 °C and 45 °C.

a Schematic representation of the LbCas12a RNP bound to target double-stranded DNA (dsDNA). The red arrows indicate the specific interactions between the LbCas12a residues and the bases of the PAM site. b Construction of a comprehensive PAM target DNA library and evaluation of PAM-mediated collateral activity of Cas12a. Using a circular plasmid as the template, the “NNNN“PAM (N = A/C/G/T) site was inserted into the former primer for PCR, generating a library of 256 target DNA (294 bp) with identical spacer. c Summary map of fluorescent kinetics for 256 PAMs in collateral activity experiments at 37 °C. d Summary map of fluorescent kinetics for 256 PAMs in collateral activity experiments at 45 °C. Each dot represents a PAM site, black for canonical PAM, colored for non-canonical PAM (red for ANNN, yellow for CNNN, green for GNNN, blue for TNNN). In these plots, each data point represents a specific PAM site: the y-axis denotes the average fluorescence intensity (defined as the maximum fluorescence value) obtained from three replicate reactions at 30 min, while the x-axis represents the time required for the fluorescence signal to reach half of that maximum value. The concentrations of dsDNA targets were 1 nM. Data are represented as mean ± standard error (n = 3 technical replicates). a.u. represents arbitrary units. Source data are provided as a Source Data file.