Jun-Jie Gogo Liu’s group developed the next-generation gene-editing tool based on RNA ribozymes
Genes carry heredity information, defining the diversity and complexity of life. Gene editing is a pivotal technology in understanding and reshaping life, playing significant roles in biological research and industry. Traditional gene editing tools, such as meganucleases, ZFNs, and TALENs, are protein-based nucleases for DNA recognition and cleavage, making it difficult to reprogram for desired editing sites. The widely used CRISPR-Cas system, an RNA-guided ribonucleoprotein enzyme, recognizes DNA through the spacer sequences in guide RNA and has excellent reprogramming capability for gene editing. However, it still faces several drawbacks, including PAM restriction, large molecular weight, and immunogenicity.
On February 1, 2024, the Jun-Jie Gogo Liu’s group at the Center for Life Sciences, published a research paper in Science titled ''Hydrolytic Endonucleolytic Ribozyme (HYER) is Programmable for Sequence-specific DNA Cleavage. '', which reports a catalytic RNA (ribozyme) – HYER (Hydrolytic Endonucleolytic Ribozyme). HYER can specifically cleave RNA and DNA substrates, inducing site-specific editing in the mammalian cell genome. Without the involvement of proteins, the recognition and cleavage of substrates are both achieved by RNA molecules, posing HYER’s potential to become the next-generation gene editing platform.
Figure 1. Three generations of gene editing tools
HYER originates from bacterial retrotransposons (GII introns), the mobile elements that can "copy and paste" (retrotranspose) within the host genome. These elements typically encode an RNA molecule and a protein with nuclease and reverse transcriptase activities, which can amplify in the host genome through retrotransposition by forming a ribonucleoprotein complex (RNP). Interestingly, through extensive bioinformatic screening, Jun-Jie Gogo Liu’s group discovered numerous ORF-less, compact group II C introns (ORF-less GII-C introns) that exist with multiple copies in bacterial genomes. This finding suggests that the sole component encoded by these introns, the RNA molecule, might have the ability to recognize and cleave target sites independently of proteins, facilitating the intron amplification within the host genome.
Figure 2. Diagram of hypothetical IEP-free GII-C intron-mediated DNA targeting.
Biochemical assays showed that these RNA molecules, around 600 nucleotides in length, exhibit significant RNA and DNA hydrolytic cleavage activity across a broad spectrum of ion concentration and temperature ranges. Therefore, researchers have named these RNA molecules Hydrolytic Endonucleolytic Ribozymes (HYERs), which is the first report of ribozymes with DNA hydrolytic cleavage capability. Notably, HYER1 and HYER2 demonstrated comparable in vitro DNA cleavage efficacy of the compact Cas12e (CasX) and Cas12l (Cas π) systems. To test the DNA cleavage capability of HYERs in cells, researchers have constructed a ccdB reporter system in E. coli, demonstrating that HYER1 and HYER2 can target and cleave the ccdB plasmid. In HEK293T cells, researchers established a frame-shifted puromycin resistance gene (puro*) reporter system. The results showed that HYER1 could induce in-frame editing of puro*, conferring cells puromycin resistance. Within the enriched cells after resistance selection, the highest editing efficiency at three target sites was 9.18%, indicating that HYER can introduce double-stranded DNA breaks and produce editing in eukaryotic cell genomes. However, within the unenriched cells, the editing efficiency ranged only from 0.09% to 0.2%, suggesting significant potential for further improvement and optimization.
Figure 3. The DNA cleavage activity of HYER
Researchers obtained high-resolution three-dimensional structure of HYER1 at 3.0 Å via cryo-electron microscopy. They discovered the homomeric structure of HYER1 and revealed the mechanism of hydrolytic DNA cleavage. HYER1 recognizes DNA substrate through an exposed single-stranded RNA region of 6 nucleotides, known as the Target Recognition Site (TRS). The DNA is captured within the catalytic core formed by domain V, where substrate hydrolysis is catalyzed through the classical two-magnesium-ion mechanism.
Figure 4. The Cryo-EM map of HYER1 and the atomic models of HYER1 catalytic core.
Based on the three-dimensional structure of HYER, researchers conducted multiple rational designs and demonstrated HYER's excellent programmability. Results showed that the sequence and length of TRS could be designed according to the substrate sequence flexibly. Insertion of a 14-nucleotide Recruiting Sequence (RS) near the TRS significantly improved HYER1's specificity and cleavage efficacy. Moreover, modifying the palindromic sequence and TRS resulted in the HYER1 heterodimer with two different TRSs, which could target different regions in double-stranded DNAsubstrates and produce customized cleavage products with 5’ overhangs, 3’ overhangs, or blunt ends.
Figure 5. Multiple rational designs improved HYER1's specificity and cleavage efficacy.
Interestingly, inspired by the "RNA World" hypothesis, researchers proposed a molecular evolutionary trajectory in which the catalytic functions of GII introns were gradually replaced by proteins. During evolution, domain IV of GII introns gradually expanded, leading to the formation of open reading frames capable of encoding short peptides. These peptides, acting as cis-elements, interacted with the intron RNA, enhancing its structural stability and catalytic activity. As the ORFs grew longer and more mature, the proteins they encoded not only stabilized the structure but also acquired DNA cleavage and reverse transcriptase activities, replacing the catalytic functions of RNA ribozymes. This work not only expands the understanding of the "RNA World" hypothesis and RNA catalytic functions, but also lays the foundation for developing novel nucleic acid manipulation platforms for gene editing and RNA editing with complete independent intellectual property rights in China.
Figure 6. The hypothetical evolution trajectory of GII intron.
Jun-Jie Gogo Liu (Associate Professor, Tsinghua) is the corresponding author of this article. Zi-Xian Liu (Ph.D candidate, Tsinghua), Shouyue Zhang (Postdoc fellow in ICSB, Tsinghua), Han-Zhou Zhu (Ph.D candidate, Tsinghua), Zhi-Hang Chen (Postdoc, Tsinghua), and Yun Yang (Ph.D candidate, Tsinghua) and Long-Qi Li (Ph.D student, Tsinghua) are co-first authors. Yun Liu (Undergraduate student, Tsinghua), Dan-Yuan Li (Ph.D candidate, Tsinghua), Ao Sun (Ph.D candidate, Tsinghua), Cheng-Ping Li (Ph.D candidate, Tsinghua), Shun-Qing Tan (Ph.D candidate, Tsinghua), Gao-Li Wang (Ph.D student, Tsinghua), Jie-Yi Shen (Ph.D student, Tsinghua), Shuai Jin (Shui Mu Postdoc fellow, Tsinghua), Caixia Gao (Research scientist, Chinese Academy of Sciences), and Yuan Lei (Ph.D candidate, Chinese Academy of Sciences) also made significant contributions to this research. Tsinghua Cryo-EM facility provided equipment and technical support for this study. This work was supported by the National Natural Science Foundation of China (Grant No. 32150018), the Ministry of Agriculture, and Tsinghua University.
Jun-Jie Gogo Liu’s group focused on nuclease mechanisms and development and application of nucleic acid manipulation tools. Employing a comprehensive approach that integrates bioinformatics, structural biology, biochemistry, and cell biology, Gogo Lab and collaborators have identified and developed various gene editing tools (Cell, 2023; Nature, 2019; Mol. Cell, 2022; Cell Res., 2023). Gogo Lab seeks postdoctoral researchers who are interested in exploring novel nucleic acid manipulation systems and RNA biology, and have backgrounds in bioinformatics and cell biology. Interested individuals can find more information on the lab's website at http://gogolab.life.tsinghua.edu.cn。
Links go to the article:
https://doi.org/10.1126/science.adh4859
Reference:
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