In a new study, professor Virginijus Šikšnys from the Life Sciences Center (VU-LSC) of Vilnius University in Lithuania and his research team cooperated with Professor Guillermo Montoya of the Novo Nordisk Foundation Protein Research Center (CPR) of the University of Copenhagen in Denmark and his research team to determine the structure of TnpB using Cryo-EM. The relevant research results were published in Nature on April 13, 2023, with the title “TnpB structure requires minimal functional core of Cas12 nuclease family”.
CRISPR Cas nuclease, such as Cas9 or Cas12, also known as gene scissors, have completely changed the field of genome editing. They can accurately edit the genome and correct pathogenic mutations. However, the size of Cas9 or Cas12 limits the use of adeno-associated viruses (AAVs) that have been used for gene therapy to deliver them to target cells.
In a previous study, the Šikšnys team reported the discovery of a new kind of programmable nuclease – TnpB -, which is related to a mobile genetic element called transposon (Nature, 2021, doi: 10.1038/s41586-021-04058-1). They confirmed that TnpB is the smallest programmable nuclease, which can be used for effective genome editing, however, its structural organization and mechanism of action are still unknown.
professor Virginijus Šikšnys said, “This new study is the result of long-term and unremitting efforts, showcasing the potential of Lithuanian scientists in the field of life sciences and their ability to become leaders in this field. This study reveals the structure and mechanism of TnpB gene scissors, laying the foundation for further targeted modification of TnpB complexes.”
In this new study, these authors used low-temperature electron microscopy to determine the ternary structure of the smallest programmable endonuclease TnpB, which, along with biochemical experimental data, explains how TnpB gene scissors accurately recognize and cleave DNA targets.
Structural studies have shown that a long RNA molecule bound to TnpB protein forms a complex three-dimensional structure, which not only helps to identify DNA targets but also controls the DNA cleavage activity of TnpB. Structural comparison and bioinformatics analysis show that TnpB is the precursor molecule of the Cas12 nuclease family and forms the structure-function core of Cas12.
As the co-corresponding author of the paper, Dr. Giedrius Sasnauskas of the Center for Life Sciences of Vilnius University, pointed out, the success of this new study depends on several factors. Sasnauskas said, “First of all, the relevant research objects, as well as the cooperation between biochemists, molecular biologists, bioinformatics scientists from the Life Science Center of Vilnius University, and colleagues from the Protein Research Center of the Novo Nordisk Foundation of Copenhagen University. But most importantly, we can use the low-temperature electron microscope available in the Life Science Center of Vilnius University to carry out this research in Lithuania.”
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Reference
Giedrius Sasnauskas et al. TnpB structure reveals minimal functional core of Cas12 nuclease family. Nature, 2023, doi:10.1038/s41586-023-05826-x.