Fig. 1: Cas9d and HEARO enzymes are dsDNA nucleases with diverse targeting ability.
From: Compact Cas9d and HEARO enzymes for genome editing discovered from uncultivated microbes
a Predicted domain architecture of Cas9d and HEARO nucleases recovered here vs. SpCas9 (not to scale). b–d Genomic context of the CRISPR-associated systems Cas9c2-MG33-33 (b), Cas9d-MG102-2 (c), and Cas9d-MG34-1 (d). The tracrRNA and CRISPR array orientations were confirmed by in vitro cleavage activity with the effector. Adaptation module genes (Cas1, Cas2, Cas4, and putative Csn2) were identified in a few cases. Environmental RNASeq reads in (d) mapped in the forward orientation to the array and intergenic region encoding a tracrRNA. Genes not associated with the described nucleases are represented by yellow arrows. e Phylogenetic protein tree of Cas9d, Cas9c2, and HEARO nucleases vs. reference sequences. The tree was inferred from a multiple sequence alignment of the shared RuvC-II/HNH/RuvC-III domains across >11,800 sequences. The Cas9c2-MG33 family of nucleases (burgundy branches) clusters with two archaeal Cas9 references, while other CRISPR-associated Cas9d (teal branches) cluster with sequences recently classified as type II-D. HEARO nucleases (lilac branches) cluster with HEARO ORF and IscB sequences (gray branches). Reference Cas9 sequences correspond to: Cj, CjCas9; Nme, NmeCas9; Sa, SaCas9; St1, St1Cas9; Fn, FnCas9; Spy, SpCas9. Reference HEARO (Ama-1-1) and IscB (KraIscB-1 and OgeuIscB1) effectors are also shown. The distance between tips was estimated as two substitutions per site (horizontal bar). f Phylogenetic clades of type II-C2 and II-D families. The clades are a zoom-in representation of the phylogenetic tree on 1E. Local support values for internal family split nodes are shown and range from 0 to 1. SeqLogo representation of consensus target motif sequences and sgRNA designs from biochemical cleavage activity assays for active Cas9c2 and Cas9d nucleases are shown. The distance between tips is estimated as 0.8 substitutions per site (horizontal bar). g Genomic context of the HEARO nuclease MG35-1 with its encoded RNA. h HEARO RNA secondary structure for two active nucleases. SeqLogo representation of consensus target motif sequences are shown.
