OMEGA systems

Tracing the origin of CRISPR-Cas

CRISPR-Cas systems have transformed genome editing and other biotechnologies; however, the broader origins and diversity of RNA-guided nucleases have largely remained unexplored. Altae-Tran et al. show that three distinct transposon-encoded proteins, IscB, IsrB, and TnpB, are naturally occurring, reprogrammable RNA-guided DNA nucleases. In addition to identifying diverse guide-encoding mechanisms, the authors elucidate the evolutionary relationship between IsrB, IscB, and CRISPR-Cas9. Overall, these newly characterized systems, called OMEGA (for obligate mobile element–guided activity) systems, are found in all domains of life and may be harnessed for biotechnology development.

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.

The first hints that OMEGA proteins might be directed by RNA came from the genes for proteins called IscBs. The IscBs are not involved in CRISPR immunity and were not known to associate with RNA, but they looked like small, DNA-cutting enzymes. The team discovered that each IscB had a small RNA encoded nearby and it directed IscB enzymes to cut specific DNA sequences. They named these RNAs “ωRNAs.”

The team’s experiments showed that two other classes of small proteins known as IsrBs and TnpBs, one of the most abundant genes in bacteria, also use ωRNAs that act as guides to direct the cleavage of DNA.

IscB, IsrB, and TnpB are found in mobile genetic elements called transposons. Graduate student Han Altae-Tran, co-first author on the paper, explains that each time these transposons move, they create a new guide RNA, allowing the enzyme they encode to cut somewhere else.

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

HAN ALTAE-TRAN

SOUMYA KANNAN

F. ESRA DEMIRCIOGLU

, RACHEL OSHIRO

, SUCHITA P. NETY

LUKE J. MCKAY

MENSUR DLAKIĆ

, WILLIAM P. INSKEEP

KIRA S. MAKAROVA

RHIANNON K. MACRAE

EUGENE V. KOONIN

, AND FENG ZHANG

OMEGA systems

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