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For a long time, biologists have sought to develop programmable methods to recombine long DNA sequences within the genome. This capability would enable researchers to insert, invert, delete, or move kilobase-sized DNA sequences to specific locations within the genome in a single step.
Researchers have been investigating recombinases and transposases capable of mediating large-scale genomic rearrangements. However, precisely targeting these enzymes to specific genomic loci has proven to be extremely challenging.
On June 26, 2024, Patrick Hsu and colleagues from Arc Institute published two research papers in Nature describing the characteristics of a recombinase guided by Bridge RNA. This tool can insert, invert, or delete long DNA sequences at specific genomic locations, thereby providing new capabilities for genome editing.
This method, which enables fundamental DNA rearrangements (insertion, inversion, or deletion) in a single step, could offer a simpler and more effective approach to genome editing compared to existing technologies. It is anticipated to achieve more precise and large-scale genome editing and mediate recombination rather than causing breakage that requires repair.
Notably, Patrick Hsu, one of the corresponding authors of these papers, is a former graduate student of Professor Zhang Feng, a pioneer in CRISPR gene editing.
BOC RNA offers specialized RNA services for the development of gene editing tools and gene therapies, providing custom RNA synthesis, including mRNA and guide RNA, to support cutting-edge research.
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The first paper is titled: "Bridge RNAs Direct Programmable Recombination of Target and Donor DNA." In this study, the research team describes a technique for using programmable recombinases in gene editing. These recombinases are guided by RNA, which acts as a "bridge" to target specific genomic sites and facilitate precise editing. This RNA bridge includes a region specifying the donor DNA sequence and another region specifying the genomic insertion site. Both regions can independently be reprogrammed to recognize and bind different DNA sequences or insertion sites, using a universal mechanism for various types of DNA rearrangements. This bridge RNA offers greater ease of modification compared to existing gene editing technologies that rely on more complex protein-DNA binding sites.
Evolution has equipped a variety of enzymes to perform genomic rearrangements and diverse tasks, facilitating the emergence and functional specialization of new genes, as well as advancing the immune system and the opportunistic spread of viruses and mobile genetic elements (MGEs). MGEs are present in all life forms and typically move through transposases, integrases, homing endonucleases, or recombinases. These enzymes often recognize DNA through protein-DNA interactions and can be broadly classified based on their sequence specificity, ranging from site-specific (e.g., Cre recombinase, Bxb1 recombinase) to semi-random (e.g., Tn5 enzyme and PiggyBac transposase).
Insertion sequence (IS) elements are among the most basic autonomous mobile genetic elements and are widespread in bacteria and archaea. Many identified IS elements use self-encoded transposases to recognize terminal inverted repeats (TIR) through protein-DNA interactions. IS elements have been classified into approximately 28 families based on their homology, structure, and transposition mechanisms. They can be roughly categorized based on the conserved catalytic residues of their encoded transposases, including DDE, DEDD, HUH transposases, and less common serine or tyrosine transposases.
The IS110 family elements are cut-and-paste type mobile genetic elements that seamlessly excise themselves from the genome and form circular intermediates. Due to this mechanism and lifecycle, IS110 transposases are more accurately described as recombinases. Although circular intermediates have been observed in other IS families, IS110 is the only family that uses the DEDD catalytic domain. IS110 elements typically lack TIR and integrate in a sequence-specific manner, often targeting repetitive sequences in microbial genomes. Although the DNA recognition and recombination mechanisms of IS110 elements are not fully understood, previous research suggests that non-coding regions flanking the recombinase ORF regulate the expression of the recombinase.
This study demonstrates that IS110 encodes both a recombinase and a non-coding bridge RNA, which specifically binds to the encoded recombinase. This bridge RNA contains two internal circular structures with nucleotide sequences that can pair with target DNA and donor DNA (i.e., the IS110 element itself). The study further shows that the target-binding and donor-binding rings can be independently reprogrammed to guide specific sequence recombination between the two DNA molecules.
This modular feature allows DNA to be inserted into genomic target sites and facilitates programmable DNA deletion and inversion. The IS110 bridge recombination system extends the diversity of nucleic acid-guided systems beyond CRISPR and RNAi, providing a unified mechanism for executing three fundamental DNA rearrangement operations—insertion, deletion, and inversion—which are crucial for genome design. Overall, the study highlights how the modular nature of bridge RNA enables the generalization of sequence-specific DNA rearrangement mechanisms, presenting a novel approach to genome editing.
The second paper is titled: "Structural Mechanism of Bridge RNA-Guided Recombination." This study uses cryo-electron microscopy to elucidate the structure of the IS110 recombinase in complex with its bridge RNA, target DNA, and donor DNA, revealing the detailed mechanism of action.
Insertion sequence (IS) elements are the simplest autonomous transposons discovered in prokaryotic genomes. The IS110 family elements encode a recombinase and a non-coding bridge RNA (Bridge RNA). This study reports cryo-EM structures of the IS110 recombinase complexed with its bridge RNA, target DNA, and donor DNA at three distinct stages of the recombination reaction cycle. Comparative analysis of these structures reveals the following processes: 1) The top strands of the target and donor DNA are cut at the active site of the complex, forming a covalent 5'-phosphoserine intermediate; 2) The cut DNA strands exchange and reconnect to form a Holliday junction intermediate; 3) The intermediate is then resolved by cutting the bottom strands.
Overall, this research reveals how the dual-specificity RNA enables IS110 recombinase to specifically target and recombine DNA, facilitating programmable DNA recombination.
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