5 April 2023

Snapshots of the smallest programmable nuclease TnpB published in Nature

Cryo-electron microscopy
Cryo-electron microscopy was used to reveal the structure of the TnpB gene scissors.

The article "TnpB structure reveals minimal functional core of Cas12 nuclease family" was published in the prestigious journal Nature.

CRISPR-Cas nucleases, such as Cas9 or Cas12, also known as gene scissors, have revolutionized the field of genome editing. They enable precise editing of genomes and correction of disease-causing mutations. However, the size of Cas9 or Cas12 limits their delivery to target cells using Adeno-associated viruses (AAV), which are already used in gene therapy.

In their previous "Nature" paper (Karvelis et al., 2021) VU LSC researchers reported the discovery of a new class of programmable nucleases called TnpBs, which are associated with mobile genetic elements called transposons. They demonstrated that TnpB is so far the smallest programmable nuclease that can be applied for efficient gene editing; however, its structural organization and mechanism remained unknown.

“The new Nature paper is a result of a consistent and long-term effort that reveals the structure and mechanism of TnpB gene scissors, which creates a basis for further targeted engineering of the TnpB complex to transform it into a therapeutic tool for the treatment of genetic disease," says Professor V. Šikšnys.

In the current study, the research team at VU LSC used cryo-electron microscopy (cryo-EM) to determine the ternary structures of the smallest programmable endonuclease, TnpB, which, along with biochemical studies, explained how TnpB gene scissors are able to precisely recognize and cut DNA targets.

Structural studies revealed that a long RNA molecule associated with the TnpB protein forms a complex three-dimensional structure that not only helps to recognize the DNA target but also controls TnpB's DNA-cutting activity. A comparison of structures and bioinformatic analysis revealed that TnpB is the precursor of the Cas12 nuclease family and forms the Cas12 structural-functional core.

As noted by one of the authors of the article, Dr. Giedrius Sasnauskas, the success of this research is a result of a cross-disciplinary collaboration of VU LSC biochemists, molecular biologists, bioinformaticians, and the Montoya group at CPR.

Read the full study TnpB structure reveals minimal functional core of Cas12 nuclease family in Nature.

Contact: Guillermo Montoya (guillermo.montoya@cpr.ku.dk)

Sasnauskas, G. Tamulaitiene, G. Druteika, A. Carabias, A. Silanskas, D. Kazlauskas, Č. Venclovas, G. Montoya, T. Karvelis, V. Siksnys. TnpB structure reveals minimal functional core of Cas12 nuclease family. Nature (2023) doi: 10.1038/s41586-023-05826-x.