RNA-programmable endonuclease systems and uses thereof
Inventors
COHNEN, Andre • Schmidt, Moritz • Coco, Wayne • GAMALINDA, Michael Biag • Gupta, Ashish • PITZLER, Christian • Richter, Florian • Tebbe, Jan • Cheng, Christopher • Takeuchi, Ryo • REISS, Caroline W.
Assignees
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Publication Number
US-12203110-B2
Publication Date
2025-01-21
Expiration Date
Abstract
Aspects of this invention, inter alia, relate to novel systems for targeting, editing or manipulating DNA in a cell, using novel synthetic RNA-guided nucleases (sRGNs). The sRGNs are derived from wildtype or parental small type II CRISPR Cas9 endonucleases.
Core Innovation
The invention relates to synthetic RNA-guided nuclease (sRGN) polypeptides comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 2. The sRGN polypeptides are used together with an RNA guide that directs activity to a target polynucleotide to enable introducing a single-stranded or a double-stranded break at a target locus.
The sRGN systems are configured to target DNA in a PAM-directed manner, and the guide RNA is described in terms of a DNA targeting segment and an sRGN-interacting second segment. The document further includes kit and composition embodiments that include an sRGN polypeptide or an encoding nucleic acid and a guide RNA configured to hybridize to a target polynucleotide.
The disclosed constructs include codon-optimized variants, specific substitutions, and sequence-listed polynucleotides and amino acid sequences. The document also describes nuclease-to-nickase conversion and abrogation by introducing nuclease activity-reducing mutations, optional fusion to nucleobase editing/deaminase polypeptides, and system delivery and implementation concepts including DNA/RNA, mRNA, AAV-encoded donor templates, liposome/lipid nanoparticle, and ribonucleoprotein (RNP) pre-complexes.
Claims Coverage
The independent claims cover three inventive features: sequence-identity-defined sRGN polypeptides, refined sRGN polypeptide variants, and a system that combines an sRGN polypeptide with a single guide RNA to induce single-stranded or double-stranded breaks in a target polynucleotide. The core claim framework is centered on an sRGN polypeptide defined by identity to SEQ ID NO: 2 and an RNA guide whose DNA targeting segment hybridizes to the target polynucleotide.
Synthetic RNA-guided nuclease polypeptide by sequence identity
A synthetic RNA-guided nuclease (sRGN) polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 2.
Engineered identity-defined sRGN polypeptide with engineered functional refinements
The sRGN polypeptide of SEQ ID NO: 2, wherein the amino acid sequence has at least 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 2, or wherein the amino acid sequence is SEQ ID NO: 2.
sgRNA-directed break induction system using an identity-defined sRGN polypeptide
A system for introducing a single-stranded or a double-stranded break in a target polynucleotide, comprising a sRGN polypeptide comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 2; and a single guide RNA (sgRNA) comprising a DNA targeting segment capable of hybridizing to a target polynucleotide, wherein the sgRNA combines with the sRGN polypeptide to induce a single-stranded or double-stranded break in the target polynucleotide.
Overall claim coverage centers on an sRGN polypeptide defined by high sequence identity to SEQ ID NO: 2 and a corresponding system where an sgRNA with a DNA targeting segment hybridizes to a target polynucleotide to induce single-stranded or double-stranded breaks, with additional refinements supported by dependent claims for delivery, packaging, formulation, donor-template insertion, and optional functional engineering features.
Stated Advantages
Enables programmable DNA targeting and induction of single-stranded or double-stranded breaks in a target polynucleotide.
Allows nuclease activity reduction (nuclease-to-nickase/abrogation) via engineered nuclease activity-reducing mutations.
Supports nucleobase editing/deaminase functionality via optional nucleobase editing/deaminase polypeptide fusion.
Supports delivery and implementation as part of a system including packaging/formulation options and kit/system configurations.
Documented Applications
Targeting/editing/modifying/manipulating target DNA at a target locus using the sRGN polypeptide and guide RNA system.
Introducing a single-stranded or double-stranded break in a target polynucleotide as part of the described system.
Inserting a donor template with a heterologous polynucleotide into a target polynucleotide as part of the described system features.
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