RNA-guided DNA integration using Tn7-like transposons
Inventors
Sternberg, Samuel Henry • Klompe, Sanne Eveline
Assignees
Columbia University in the City of New York
Publication Number
US-12331292-B2
Publication Date
2025-06-17
Expiration Date
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Abstract
In certain embodiments, the present systems and methods use Tn7-like transposons that encode CRISPR-Cas systems for programmable, RNA-guided DNA integration. For example, the CRISPR-Cas machinery directs the Tn7 transposon-associated proteins to integrate DNA downstream of a target site (e.g., a genomic target site) recognized by a guide RNA (gRNA).
Core Innovation
In certain embodiments, the present systems and methods use Tn7-like transposons that encode CRISPR-Cas systems for programmable, RNA-guided DNA integration, where the CRISPR-Cas machinery directs the Tn7 transposon-associated proteins to integrate DNA downstream of a target site recognized by a guide RNA. The present systems and methods provide for RNA-guided DNA integration comprising an engineered CRISPR-Cas system derived from a Type I CRISPR-Cas system that includes a guide RNA specific for a target site, together with an engineered transposon system derived from a Tn7-like transposon system that comprises TnsA, TnsB, TnsC and TnsD/TniQ, and a donor DNA comprising a cargo nucleic acid flanked by transposon end sequences.
The background describes limitations and risks associated with the use of programmable nucleases (e.g., CRISPR-Cas9) for insertion of large gene cargos into eukaryotic genomes, including the requirement for double-strand breaks (DSBs), reliance on homology-directed repair (HDR), low HDR efficiencies in many cell types, hazards from DSBs such as off-target mutations and DNA damage responses, and the absence of HDR machinery in post-mitotic cells. The present disclosure states that the RNA-guided DNA integration systems and methods obviate the need to introduce DSBs, and thus preclude the above hazards, and that the systems have significant utility in genetic engineering, including mammalian cell genome engineering.
The disclosure further describes that the engineered CRISPR-Cas system may comprise Cas6, Cas7, Cas5 and Cas8 (optionally as a Cas8-Cas5 fusion) and the engineered transposon system may be derived from Vibrio cholerae Tn6677 or other specified bacteria, and that integration occurs proximal to a target site and can be achieved without homologous recombination. Experimental constructs, expression strategies, delivery approaches, and assays for reconstituting RNA-guided DNA integration are described in detail in the specification [procedural detail omitted for safety].
Claims Coverage
Overview of the claim coverage. One independent claim was provided and five main inventive features are extracted.
Engineered type I CRISPR-Cas system
An engineered CRISPR-Cas system derived from a Type I CRISPR-Cas system comprising at least one Cas protein and a guide RNA (gRNA) specific for a target site, wherein the CRISPR-Cas system binds to the target site.
Engineered Tn7-like transposon system
An engineered transposon system derived from a Tn7-like transposon system comprising transposition proteins including TnsA, TnsB, and TnsC, and optionally TnsD and/or TniQ, that integrates donor DNA proximal to a target site.
Donor DNA flanked by transposon end sequences
A donor sequence comprising a cargo nucleic acid sequence flanked by first and second transposon end sequences that is provided for integration by the engineered transposon system.
RNA-guided integration on the PAM-distal side
Integration of the donor sequence into the target nucleic acid occurs on the PAM-distal side of the target site specified by the guide RNA.
Configurable vector delivery and species-matched systems
The engineered CRISPR-Cas system and engineered transposon system can be provided on the same or different vector(s), and the engineered transposon system and engineered CRISPR-Cas system are derived from the same species.
The independent claim covers a programmable RNA-guided DNA integration method combining an engineered Type I CRISPR-Cas effector (at least one Cas protein plus gRNA), an engineered Tn7-like transposon system (TnsA/TnsB/TnsC and optional TnsD/TniQ), and a donor DNA flanked by transposon ends, delivered on the same or separate vectors, with integration occurring on the PAM-distal side of the target site and with the CRISPR and transposon components derived from the same species.
Stated Advantages
Obviates the need to introduce double-strand breaks (DSBs) and thus precludes hazards associated with DSBs.
Enables RNA-guided DNA integration without homologous recombination (HDR).
Provides significant utility in genetic engineering, including mammalian cell genome engineering.
Permits targeted, programmable integration of donor DNA downstream of a gRNA-specified target site, avoiding random viral integration and associated insertional mutagenesis.
Allows DNA integration in cell types with low or absent HDR machinery, including post-mitotic and non-dividing cells.
Accommodates integration of large donor DNA cargos (e.g., donors of at least 2 kb and larger sizes are described).
Documented Applications
Genetic engineering and genome engineering in a variety of cells, including mammalian, avian, plant, fish, and bacterial cells, using programmable RNA-guided DNA integration.
Insertion of donor DNA into eukaryotic genomes for therapeutic or experimental purposes, including applications in mammalian cells and human cells.
Gene therapy and treatment methods involving administration of compositions comprising engineered transposon-encoded CRISPR-Cas systems to a subject for integration of therapeutic sequences, with diseases explicitly listed including cancer, Duchenne muscular dystrophy (DMD), sickle cell disease (SCD), β-thalassemia, and hereditary tyrosinemia type I (HT1).
Plant genome engineering to confer or modify agronomic traits (examples explicitly described include grain number, grain size, grain weight, panicle size, tiller number, fragrance, nutritional value, shelf life, lycopene content, starch content, lower gluten content, reduced toxin levels, asexual propagation, improved haploid breeding, shortened growth time, herbicide tolerance, drought tolerance, male sterility, insect resistance, abiotic stress tolerance, modified fatty acid metabolism, modified carbohydrate metabolism, modified seed yield, modified oil percent, modified protein percent, and resistance to bacterial, fungal, or viral disease).
Genetic modification of animal cells and generation of genetically modified non-human animals, tissues, organs, or cell populations, including applications in livestock and laboratory animals.
Inactivation of microbial genes, including antibiotic resistance genes, virulence genes, or metabolic genes, by insertion of donor DNA into microbial genes.
Microbiome engineering and delivery via bacterial conjugation, including passing transposon-encoded CRISPR-Cas systems to recipient bacterial cells by conjugation.
Targeted DNA enrichment and library preparation for next-generation sequencing (NGS) by appending transposon end sequences or adaptors to input DNA via RNA-guided DNA integration for downstream PCR amplification and sequencing.
Development of kits and compositions comprising one or more vectors encoding engineered CRISPR-Cas and Tn7-like transposon systems, and associated components such as infusion devices, solution bags, buffers, control plasmids, and sequencing primers.
Multiplexed insertion and pooled guide RNA library strategies for high-throughput screening or multiplexed genome engineering, including arrayed or pooled gRNA libraries encoded on arrays or within donor cargos.
Use in ex vivo cell modification workflows for cell therapy, including potential applications such as CAR-T cell engineering where defined genomic insertion sites are desirable (described as therapeutic and experimental utility).
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