Gene editing components, systems, and methods of use
Inventors
Gasiunas, Giedrius • LADHA, Alim • Presnyak, Vladimir • Jayaraman, Muthusamy • Narayanan, Elisabeth
Assignees
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Publication Number
US-12264328-B2
Publication Date
2025-04-01
Expiration Date
Abstract
The present disclosure provides methods and compositions comprising Cas TypeV programmable nucleases and lipid nanoparticles capable of delivering the Cas TypeV programmable nucleases and genome editing systems comprising same.
Core Innovation
The disclosure describes isolated or recombinant polynucleotides encoding polypeptides that are at least 95% identical to SEQ ID NO: 585 and comprise arginine (R) at position 169. The polypeptides belong to a Cas12a-class genome editor family with RNA-guided activity and named domain architectures including WED, REC, PI, BH, RuvC, and NUC. The document further describes Cas12a/Cpf1-based editing systems, alternative RNA-guided nucleases, and engineered variants.
The guide RNA architecture includes a direct repeat and a targeting guide, with PAM-recognition constraints including TTTN, TTV, and relaxed PAM aspects. The disclosure also describes crRNA-only operation for Cas12a/Cpf1, nickase and dead Cas configurations, base editing using deaminases, prime editing components including pegRNA and reverse transcriptase fusion, and donor polynucleotides for repair. Related systems include Cas12b/C2c1 and engineered FokI-dCas9 fusions.
Delivery and implementation are described with viral and non-viral vectors, including lipid nanoparticles having ionizable lipids, structural lipids, PEGylated lipids, and phospholipids, as well as targeting moieties and host cell categories. The document further describes computational prediction of Cas12a variants, sequence-identity and mutagenesis-based variant selection, and RNA payload architectures including linear and circular RNA, poly(A)-tail modifications, miRNA binding elements, and options for chemically modified nucleotides.
Claims Coverage
The provided independent claims cover two inventions: an isolated or recombinant polynucleotide, and a polypeptide or isolated polypeptide. Across the claim set, the inventive features center on a polypeptide at least 95% identical to SEQ ID NO: 585 with arginine (R) at position 169, with dependent claims further referring to domain content, reverse transcriptase fusions, LNP component classes, and host cell categories.
Polynucleotide encoding SEQ ID NO: 585 with arginine at position 169
An isolated or recombinant polynucleotide comprising a nucleic acid sequence that encodes a polypeptide at least 95% identical to SEQ ID NO: 585, wherein the polypeptide comprises arginine (R) at position 169.
Polypeptide comprising SEQ ID NO: 585 with arginine at position 169
A polypeptide or an isolated polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 585 and comprising arginine (R) at position 169.
Nuclease-related domain architecture in the encoded polypeptide
An isolated or recombinant nucleic acid sequence that encodes a polypeptide comprising named nuclease-related domains, including WED, REC, PI, BH, and RuvC domains.
Operable fusion to one or more reverse transcriptases
An isolated or recombinant nucleic acid sequence operably fused to a nucleic acid sequence encoding one or more reverse transcriptases, including M-MLV reverse transcriptase.
LNP vector composition with specified lipid classes
A vector comprising an LNP that includes one or more ionizable lipids, one or more structural lipids, one or more PEGylated lipids, and one or more phospholipids.
Isolated host cell category
An isolated host cell comprising one of a prokaryotic cell, a mammalian cell, or a synthetic cell.
Overall, the claim coverage centers on sequence-defined Cas12a polynucleotides and polypeptides that are at least 95% identical to SEQ ID NO: 585 while containing arginine (R) at position 169. Dependent features further specify nuclease-related domains, reverse transcriptase fusions, LNP-based vectors, and host cell categories.
Stated Advantages
Reduced off-target effects relative to Cas9/SpCas9.
Enhanced efficiency/precision.
Improved stability/half-life/persistence.
Reduced immunogenicity.
Documented Applications
Genome editing.
Base editing using deaminase fusions.
Prime editing using pegRNA and reverse transcriptase fusion.
Repair using donor polynucleotides for repair.
Delivery of genome editing systems using viral vectors and non-viral vectors including lipid nanoparticles (LNPs) with defined lipid classes and targeting moieties.
Therapeutic, diagnostic, prognostic, and molecular recording concepts using CRISPR protospacer-based systems.
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