Gene editing is an important biotechnology that can help people better understand and modify life. Over the past few decades, scientists have been working hard to develop gene-editing tools to achieve precise transgenesis and genetic modification. However, traditional gene editing tools such as giant nucleases, ZFNS, and TALENS use proteins to recognize and cleave DNA, and reprogramming of editing sites is difficult. Although the widely used CRISPR-Cas system has good editing ability, there are still many problems, such as PAM sequence limitation, large molecular weight, protein immunogenicity, etc. As a result, researchers are always looking for new gene editing tools to improve editing efficiency and accuracy.
Recently, Junjie Liu's research group at the School of Life Sciences of Tsinghua University has made an important breakthrough. They have blazed new trails in RNase-based gene editing research with the discovery of a catalytic RNA called Hyer (hydrolyzed endonuclease). Unlike traditional gene editing tools, HYER can specifically cleave RNA and DNA to achieve site-specific editing of the mammalian cell genome. Compared with the CRISPR-Cas system, HYER does not require protein involvement, and its substrate recognition and cleavage are completely autonomously completed by RNA molecules. The results of this study are expected to be a new generation of gene editing chassis tools after CRISPR.
(1) Limitations of meganucleases, ZFNS, and TALENS
Meganucleases, ZFNS, and TALENS are among the first gene editing tools to be developed in history. They achieve their editing function through the specific binding of proteins to DNA. However, the editing site reprogramming of these tools is relatively difficult, limiting the scope of their application.
(2) Breakthrough in the CRISPR-Cas system
The CRISPR-Cas system is one of the most commonly used gene editing tools today. It achieves specific cleavage of DNA through RNA-guided protein nucleases. Compared with previous tools, the CRISPR-Cas system has better editing site reprogramming capabilities. However, it still has some problems, such as PAM sequence limitation, large molecular weight, protein immunogenicity, etc.
(3) Discovery and characteristics of HYER
Hyer is derived from the bacterial reverse poson, a mobile element that can be "copied and pasted" on the host genome. Through extensive bioinformatics screening, Junjie Liu's group found that there are many compact class II C-type introns in the bacterial genome that do not code for proteins. These intron-encoded, unique component RNA molecules, may have protein-independent cleavage capabilities and can autonomously recognize and cleavage substrates through RNA molecules. In experiments, the researchers found that these RNA molecules had significant RNA and DNA hydrolytic cleavage activity over a broad spectrum of ion concentrations and temperatures. Therefore, the researchers named these RNA molecules Hyer (hydrolyzed endonuclease).
(1) Hyer's DNA cleavage ability
The researchers found that HYER can perform specific cleavages of DNA and achieve targeted cleavage of plasmids carrying the CCDB virulence gene. In eukaryotic cell genomes, hyer can introduce double-strand breaks and produce editing effects. However, the editing efficiency of HYER needs to be further improved and optimized.
(2) Programmability of HYER
Based on the three-dimensional structure and rational design of the hyer, the researchers proved that the hyer has good programmability. They can flexibly design the sequence and length of the hyer according to the substrate sequence, and realize different styles and lengths of cleavage products by modifying the palindromic sequence and the recognition sequence.
(3) The relationship between HYER and the RNA world hypothesis
Researchers believe that the discovery and evolution of Hyer supports the RNA world hypothesis. In the process of evolution, the RNA of the second type of intron is gradually replaced by protein, forming introns with protein catalytic functions. This discovery not only expands the understanding of the RNA world hypothesis and the catalytic function of RNA, but also lays the foundation for the creation of a new nucleic acid manipulation chassis tool with independent intellectual property rights.
Junjie Liu's research group at the School of Life Sciences of Tsinghua University has discovered a catalytic RNA with DNA cleavage ability, named HYER. Unlike traditional gene editing tools, Hyer can specifically cleave RNA and DNA and enable genome editing. At present, the editing efficiency of HYER needs to be improved and optimized, but the results of this study provide a new direction for the development and application of a new generation of gene editing tools. In addition, Hyer's discovery has expanded the understanding of the RNA world and the catalytic function of RNA. In the future, scientists can further explore the potential of HYER and improve its editing capabilities, as well as delve into the origin and evolution of the RNA world. It is hoped that through these efforts, more breakthroughs and innovations can be brought to the development and application of gene editing technology, and contribute to the progress of human life sciences.