Gene editing is one of the latest breakthroughs in biology. The well-known CRISPR-Cas gene editing system confers immunity against foreign DNA to prokaryotes (organisms lacking a cell nucleus). Since the discovery of CRISPR gene editing technology, scientists have revealed how CRISPR-cas proteins evolved from their precursors. This knowledge will help them develop other small new genome editing tools for gene therapy.
At the University of Tokyo, Professor Osamu Nureki's team worked to identify the structure and function of proteins involved in genome editing. In a recent study by the team, they discovered the 3D structure of protein TnpB, a possible precursor to the CRISPR-Cas12 enzyme. Their findings were published in Nature.
Previous research has shown that the TnpB protein may act like a pair of molecular scissors, cutting DNA with the help of a special type of non-coding RNA called omega RNA. But how RNA-guided DNA cleavage works, and its evolutionary relationship to the Cas12 enzyme, was unclear, prompting research from Nureki's lab. The first and most critical step in their understanding was to reveal the protein's structure.
To determine the three-dimensional structure of TnpB, the researchers extracted the TnpB protein from a bacterium called Deinococcus radiodurans and used cryo-electron microscopy. In cryo-electron microscopy, a protein sample is cooled to -196°C using liquid nitrogen and illuminated with an electron beam, revealing the protein's 3D structure.
The team found that the omega RNA in TnpB has a unique pseudoknot shape, similar to the guide RNA for the Cas12 enzyme. The study also revealed how TnpB recognizes omega RNAs and cleaves target DNA. When they compared the structure of this protein to the Cas12 enzyme, they learned two possible ways that TnpB might have evolved into a CRISPR-Cas12 enzyme.
"Our findings provide mechanistic insights into TnpB function and advance our understanding of the evolution of TnpB proteins to CRISPR-Cas12 effectors,” said Ryoya Nakagawa, one of the first authors of the research paper. He added that, “In the future, we will explore the potential application of tnpb-based gene editing technology.”
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