Fig. 5: Biochemical characterization of Casλ homologs.

a Structure of pre-crRNA substrates consists of a hairpin formed by a direct repeat (DR) sequence followed by a 20 bp spacer. Diamonds symbols indicate directed repeats (DR), and colored oval squares indicate different spacers. b Representative gel of Casλ-mediated pre-crRNA cleavage by six Casλ homologs after 60 min incubation with 5′-FAM labeled pre-crRNA substrates. c Pipeline used to detect dsDNA cleavage and associated PAM recognition by in vitro DNA cleavage assay. Casλ RNP complexes cleave a 5′ PAM library PCR product in vitro, and the uncut part was captured via PCR and subjected to Illumina deep sequencing. Gray rectangles indicate 5′ PAM library PCR products, with blue blocks representing spacers and colored short blocks denoting various PAM motifs. Dark green and dark blue squares represent different barcodes on the sequencing adapters. d Six Casλ homologs cleaved dsDNA in vitro at 37 °C for 1 h. A 500 bp PCR product was cleaved into two 250 bp products. e Analysis of Illumina deep sequencing data showing that the presumed PAM of EphcCasλ was TTR. The weblogo of the presumed PAM that supported target recognition and cleavage was generated using WebLogo (Thymine is colored red, Adenine green, Cytosine blue, and Guanine yellow). Source data are provided as a Source Data file. f EphcCasλ and Casλ1 cleaved TTA/TTG dsDNA in vitro at 37 °C for 3 h. PAM was confirmed to be TTA/TTG. g Trans-cleavage assay conducted with the ssDNA-FAM/BHQ reporter. This reporter is a ssDNA oligonucleotide labeled with a fluorophore (FAM) at one end and a quencher (BHQ) at the other. Initially, fluorescence from FAM is quenched by BHQ due to their proximity (top left). Upon recognizing and binding to a target DNA sequence, the Casλ nuclease becomes activated and can non-specifically cleave nearby ssDNA, including the reporter. As a result, the fluorophore (FAM) is separated from the quencher (BHQ), leading to the emission of fluorescence (bottom right). The activated Casλ is shown in pink, the target DNA sequence in black, the PAM motif in yellow, and the guide RNA in blue. h EphcCasλ exhibited trans-cleavage of DNA at 30 °C, 37 °C, and 44 °C when the PAM was TTA/G. The experiments are shown in (b, d, f) are representative of three independent experiments with similar results.