US20260035682
2026-02-05
Chemistry; metallurgy
C12N9/22
RNA-guided nucleases (RGNs) are advanced tools in molecular biology, offering precise methods for genome editing. These compositions, including CRISPR RNAs, trans-activating CRISPR RNAs, and guide RNAs, enable targeted binding and modification of DNA sequences. By utilizing vectors and host cells that carry these nucleic acid molecules, RGNs can effectively cleave or alter target sequences, visualize specific DNA regions, and adjust gene expression.
The development of targeted genome editing has evolved from earlier methods involving engineered nucleases like meganucleases and TALENs, which required labor-intensive creation of specific DNA-binding domains. RNA-guided nucleases, particularly CRISPR-associated proteins, simplify this process by using guide RNAs to direct nucleases to specific DNA sequences. This approach is more efficient and cost-effective, allowing for the introduction of mutations or the insertion of new DNA through mechanisms like non-homologous end joining or homology-directed repair.
The core components of the RGN system include RNA-guided nuclease polypeptides and guide RNAs. These elements work together to bind and modify target DNA sequences. The system can introduce double-stranded breaks, single-stranded breaks, or perform base editing by incorporating additional proteins like deaminases. The guide RNAs are designed to hybridize with specific DNA sequences, ensuring precise targeting and modification.
RGNs have broad applications in gene editing, from basic research to therapeutic interventions. They allow for precise modifications of genetic material, facilitating studies on gene function and the development of treatments for genetic disorders. The ability to introduce specific mutations or insert new genetic material opens up possibilities for correcting genetic defects and enhancing desirable traits in organisms.
The invention includes nucleic acid molecules encoding RGN polypeptides with high sequence identity to specific sequences, ensuring effective binding and modification capabilities. These polypeptides can be linked to various editing proteins, such as DNA polymerases or deaminases, to expand their functionality. The system also includes vectors carrying these components, enabling easy delivery and expression in target cells. The guide RNAs are carefully designed to ensure compatibility with the RGN polypeptides, maximizing the efficiency and specificity of the editing process.