US20260117213
2026-04-30
Chemistry; metallurgy
C12N9/22
The patent application discusses compositions and methods for targeting specific DNA sequences using RNA-guided nucleases (RGNs). These compositions are useful for cleaving or modifying target sequences, detecting them, and altering gene expression. The system includes RGN polypeptides, CRISPR RNAs (crRNAs), trans-activating CRISPR RNAs (tracrRNAs), guide RNAs (gRNAs), and the nucleic acid molecules that encode them. Additionally, vectors and host cells containing these nucleic acids are part of the invention. The RGN system is designed to bind target sequences with the help of guide RNAs.
The invention includes a nucleic acid molecule encoding an RGN polypeptide, which can bind a target DNA sequence in an RNA-guided, sequence-specific manner. This binding is facilitated by a guide RNA capable of hybridizing with the target. The RGN polypeptide can have a sequence identity of at least 90% to specified sequences and is capable of cleaving the target DNA, generating either double-stranded or single-stranded breaks. Some RGN polypeptides are designed to be nuclease inactive or act as nickases.
The RGN polypeptides may be linked to various functional components such as prime editing polypeptides or base-editing polypeptides. Prime editing polypeptides could include DNA polymerase or reverse transcriptase, while base-editing polypeptides could include deaminases like cytosine or adenine deaminase. These components enhance the versatility of the RGN system, allowing for precise genetic modifications.
Vectors carrying the nucleic acid molecules are also included in the invention. These vectors can further include sequences encoding gRNAs that hybridize with the target DNA. The guide RNAs are designed to work with specific RGN polypeptides, ensuring targeted and efficient DNA editing. This system can be used for a variety of applications, from basic research to therapeutic interventions, by enabling precise genome editing.
The guide RNAs are carefully designed to include CRISPR repeat sequences and tracrRNAs with high sequence identity to specified sequences. This ensures that the RGN polypeptides can effectively bind and edit specific DNA targets. The invention's design allows for flexibility and specificity in targeting different genomic sequences, enhancing the potential for widespread use in various fields of molecular biology and gene editing.