The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases

TLDR

IscB proteins are putative nucleases encoded in IS200/IS605 transposons and are likely ancestors of Cas9, but their functions and RNA interactions remain uncharacterized. The study aims to reconstruct the evolution of CRISPR‑Cas9 systems from IS200/IS605 transposons using evolutionary analysis, RNA sequencing, and biochemical experiments. The authors employed evolutionary analysis, RNA sequencing, and biochemical assays to trace the emergence of RNA‑guided endonucleases from these transposons. They discovered that IscB and TnpB, both encoded by IS200/IS605 transposons, perform RNA‑guided cleavage of double‑stranded DNA, can be used for genome editing in human cells, and together define a widespread class of transposon‑encoded RNA‑guided nucleases named OMEGA with strong biotechnological potential.

Abstract

IscB proteins are putative nucleases encoded in a distinct family of IS200/IS605 transposons and are likely ancestors of the RNA-guided endonuclease Cas9, but the functions of IscB and its interactions with any RNA remain uncharacterized. Using evolutionary analysis, RNA sequencing, and biochemical experiments, we reconstructed the evolution of CRISPR-Cas9 systems from IS200/IS605 transposons. We found that IscB uses a single noncoding RNA for RNA-guided cleavage of double-stranded DNA and can be harnessed for genome editing in human cells. We also demonstrate the RNA-guided nuclease activity of TnpB, another IS200/IS605 transposon-encoded protein and the likely ancestor of Cas12 endonucleases. This work reveals a widespread class of transposon-encoded RNA-guided nucleases, which we name OMEGA (obligate mobile element–guided activity), with strong potential for developing as biotechnologies.