New Class of Transposon-Encoded RNA-Guided Nucleases May Add to Genome Editing Toolbox

NEW YORK – A team of US researchers led by the Broad Institute’s Feng Zhang has discovered a new class of transposon-encoded RNA-guided DNA nucleases, which they said could be used for genome editing in human cells and hold potential for biotechnology.

In a paper published on Thursday in Science, the researchers wrote that the IscB proteins — which are the likely ancestors of the RNA-guided endonuclease Cas9 — are putative nucleases encoded in a distinct family of IS200/IS605 transposons. Using evolutionary analysis, RNA-seq, and biochemical experiments, they reconstructed the evolution of CRISPR-Cas9 systems from IS200/IS605 transposons and showed that IscB utilized a single non-coding RNA for RNA-guided cleavage of double-stranded DNA.

The researchers also experimented with the RNA-guided nuclease activity of TnpB, another IS200/605 transposon-encoded protein and the likely ancestor of Cas12 endonucleases. Overall, they said, this work revealed a widespread class of transposon-encoded RNA-guided nucleases, which they named OMEGA, for Obligate Mobile Element Guided Activity.

IscB is about 400 amino acids long and has an architecture similar to that of Cas9 — it contains an RuvC endonuclease domain split by the insertion of a bridge helix as well as an HNH endonuclease domain. When the researchers performed a comprehensive search for proteins containing an HNH or a split RuvC endonuclease domain, they found that Cas9 and IscB were the only proteins that contained both domains. Clustering and phylogenetic analyses of the combined RuvC, BH, and HNH domains strongly suggested that all extant Cas9s descended from a single ancestral IscB.

Using a previously established protospacer adjacent motif (PAM)-discovery assay, they further observed that CRISPR-associated IscBs are reprogrammable RNA-guided nucleases. Additional experiments showed that IscB functionally associated with CRISPR at least once, and likely on additional occasions, suggesting that IscB systems more generally shared a core ancestral ncRNA gene that was prone to evolving into a CRISPR array or a separate trans-acting tracrRNA.

The researchers also investigated the evolutionary relationships between IscB, Cas9, and other homologous proteins to gain a broader insight into the evolution of RNA-guided mechanisms. In searching for proteins containing split RuvC domains, they detected another group of shorter IscB homologs that were about 350 amino acids long and were also encoded in IS200/605 superfamily transposons. They renamed these proteins IsrB (Insertion sequence RuvC-like OrfB).

In addition to IscB and IsrB, they further identified a family of even smaller (about 180 amino acids) proteins that only contained the PLMP domain and HNH domain but no RuvC domain, which they named IshB (Insertion sequence HNH-like OrfB).

In investigating the relationships between these proteins, they found that IsrB, IscB, and Cas9 formed distinct, strongly supported clades, suggesting that each of these nucleases originated from a unique evolutionary event. Additionally, they were able to identify two distinct groups of Cas9s. The first was a new subtype called II-D — a group of relatively small Cas9s about 700 amino acids long that are not associated with any other known cas genes. The second is a distinct clade branching from within the II-C subtype, which includes exceptionally large Cas9s (more than 1,700 amino acids) that are associated with tnpA.

“Through the exploration of Cas9 evolution, we discovered the programmable RNA-guided mechanism of three highly abundant but previously uncharacterized transposon-encoded nucleases: IscB, IsrB, and TnpB, which we collectively refer to as OMEGA … because the mobile element localization and movement likely determines the identity of their guides,” the authors concluded. “Although the biological functions of [OMEGA] systems remain unknown, several hypotheses are compatible with the available evidence, including roles in facilitating TnpA-catalyzed, RNA-guided transposition, or acting as a toxin.”

They further noted that the TnpB family is far more abundant and diverse than the IscB family, and that TnpBs might represent a vast diversity of RNA-guided mechanisms present not only in prokaryotes, but also in eukaryotes.

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