Fig. 6.

A family of putative non-Tn7 CASTs. (A) The defining features of this family of systems are an Rpn family (PDDEXK domain-containing) nuclease/transposase near a nuclease-dead Cas12 or a Type I-E Cascade complex. The operon is enriched for nucleic acid–processing proteins. We also observed homing spacers (magenta, black arrows) and short inverted repeats (blue) in some systems. (B) Multiple sequence alignment of Rpn proteins with the putative transposases from these systems. Residues critical for DNA cleavage in the PDDEXK domain are highlighted in red. The D165A mutant in RpnA more than doubles recombination in vivo; this aspartic acid is highlighted in red below the transposase_31 domain. (C) A schematic of an atypical homing spacer and its DNA target. The PAM is highlighted. (D) Multiple sequence alignment of nuclease-active Cas12a and putative CAST Cas12 proteins. Putative CAST Cas12 proteins retain the conserved residues in the WED domain that are essential for crRNA processing but lack an aspartic residue in the RuvC domain that is essential for DNA cleavage.