Fig. 2: Target DNA recognition.

a Recognition of the target DNA. The PAM duplex is bound to the cleft formed by the WED and REC domains. PAM upstream DNA exhibits additional interactions with the WED domain. b Detailed interactions of RdCas12n with the PAM duplex and PAM upstream DNA. c–f Major interactions between the PAM duplex and RdCas12n. Hydrogen bonding and electrostatic interactions are shown as blue dashed lines, and van der Waals interactions are shown as green dashed lines. The residues that interact with the nucleic acids through their main chains are shown in parentheses. g In vitro DNA cleavage activities of WT-RdCas12n and different PAM-recognition mutants. A linearized target DNA segment of 2.2-kb, comprising a 20-nucleotide target sequence and at a concentration of 10 nM, was subjected to incubation with the RNP complex at a concentration of 250 nM, at 37 °C across various time durations, as depicted by the gradient gray labels. Data are represented as mean ± SD (n = 3 biologically independent samples). Source data are provided as a Source Data file. h–i Major interactions between the PAM upstream DNA and RdCas12n. j Location of residues involved in PAM upstream DNA interactions. k Schematic of the molecular design for extra-WED region deletion mutants. l In vitro DNA cleavage activities of WT-RdCas12n, the PAM upstream DNA recognition mutants, and the extra-WED domain deletion mutants. Data are represented as mean ± SD (n = 3 biologically independent samples). Source data are provided as a Source Data file. m, n Guide:TS DNA recognition. Hydrogen bonding and electrostatic interactions are shown as blue dashed lines. The residues that interact with the nucleic acids through their main chains are shown in parentheses.