Gene editing using homology-independent universal genome engineering technology
Inventors
Gao, Yudong • Soderling, Scott
Assignees
Publication Number
US-12325855-B2
Publication Date
2025-06-10
Expiration Date
2039-02-15
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Abstract
Disclosed herein are the genetic constructs for a Homology-Independent Universal Genome Editing (HiUGE) system and methods of using said HiUGE system for genome editing. The invention relates to compositions comprising gRNA polynucleotides, insert polynucleotides, and a CRISPR-based nuclease or polynucleotide encoding a CRISPR-based nuclease.
Core Innovation
The invention is directed to a Homology-Independent Universal Genome Engineering (HiUGE) system for gene editing a subject genome. This HiUGE system includes a modular combination of a CRISPR-based nuclease (or nucleic acid sequence encoding the nuclease), a HiUGE vector with donor recognition sequences (DRS) flanking an insert, a HiUGE vector-specific gRNA (HD-gRNA) targeting the DRS, and a gene-specific vector supplying a target gene-specific gRNA (GS-gRNA). The HD-gRNA directs the CRISPR-based nuclease to cleave only the donor vector (and not the genome), releasing the insert for targeted integration at a site specifically cleaved in the genome by the GS-gRNA. This integration occurs via non-homologous end joining (NHEJ), allowing efficient and precise editing.
The background identifies a persistent need for enabling accurate, scalable, and flexible genome editing for purposes such as protein labeling, screening, and functional genomics. Previous methods, including RNAi, gene knockout platforms, and CRISPR-based HDR, have faced challenges such as inefficiency, labor intensity, species-specific limitations, and technical hurdles in generating custom payload vectors for each target gene. Other approaches, such as HITI and SLENDR, require gene- and/or species-specific donor vectors, limiting their scalability for genome-wide applications. Additionally, antibody-based labeling is hindered by specificity and reproducibility problems.
The HiUGE system overcomes these problems by employing donor vectors that are universally compatible with various gene targets, obviating the need for custom vectors for each gene. The use of gRNA pairs (one for the genome, one for the donor) enables modularity, high-throughput, and species-independent application. The inclusion of intein-mediated split versions of the nuclease furthers delivery flexibility. As described, the HiUGE vectors can carry diverse payloads—including epitope tags, fluorescent proteins, enzymes, and trafficking tags—and are deliverable via viral or non-viral methods. This system thus enables homology-independent, universal, and efficient genome engineering without the need for gene-specific donor constructs.
Claims Coverage
The independent claims principally protect the structure and functionality of a Homology-Independent Universal Genome Engineering (HiUGE) system, specifying key genetic components and their interactions for genome editing.
Split-intein CRISPR-based nuclease assembly for genome editing
The system comprises a HiUGE vector containing: - A first polynucleotide sequence encoding at least one insert (payload). - At least one donor recognition sequence (DRS) flanking each side of the insert, where each DRS contains a cleavage site for the CRISPR-based nuclease. - A second polynucleotide sequence encoding a HiUGE vector specific gRNA (HD-gRNA), which targets the CRISPR-based nuclease to the DRS and is inert with respect to the subject genome. - A third polynucleotide sequence encoding a first portion of a split CRISPR-based nuclease with a first split-intein. Additionally, a gene-specific vector includes: - A fourth polynucleotide sequence encoding a second portion of the CRISPR-based nuclease containing a second split-intein, complementary to the first split-intein. This allows reconstitution of the CRISPR-based nuclease after protein splicing. - A fifth polynucleotide sequence encoding a gene-specific gRNA (GS-gRNA) directing the CRISPR-based nuclease to a specific sequence within the subject genome. These elements together enable precise insertion of the payload into the genome in a homology-independent manner.
Overall, the claims emphasize the modular HiUGE system combining universal donor vectors, genome- and vector-specific gRNAs, and a split-intein CRISPR-based nuclease—enabling homology-independent genome engineering through programmable, robust, and scalable insertions.
Stated Advantages
Eliminates the need to create individual gene-specific donor vectors for every genome editing application.
Simplifies and accelerates genome-wide and high-throughput genome editing, screening, and protein labeling.
Enables precise, robust, and scalable gene editing in various cell types and across different species due to its universal design.
Reduces off-target effects by using a HiUGE vector-specific gRNA that does not target the subject genome.
Facilitates flexible and modular insertion of diverse payloads, including epitope tags, fluorescent proteins, enzymes, and trafficking tags.
Allows high efficiency and specificity through the use of dual-gRNA targeting and non-homology-based DNA repair.
Enhances the versatility of delivery methods, including compatibility with viral (AAV, lentivirus) and non-viral vectors.
Supports applications in both dividing and non-dividing cells, and in vivo as well as in vitro.
Obviates problems with traditional antibody-based protein labeling by enabling direct insertion of tags at the genetic level.
Documented Applications
Genome-wide protein labeling in cells and tissues using universal epitope tags, fluorescent proteins, enzymes, or cellular trafficking signals.
High-throughput screening of gene or protein function, including expression marking and disruption of protein expression.
In situ proximity-dependent biotinylation for proteomics studies and mapping protein interactions.
Protein truncation for structure-function studies and targeted depletion of endogenous proteins.
Live imaging of proteins in cells or tissues by inserting fluorescent protein payloads.
Subcellular relocation or trapping of endogenous proteins using localization signal inserts.
Labeling proteins in specific cells or neural circuits using viral vector delivery (AAV, lentivirus) in vivo.
Use in primary cell cultures, animal models (including mice), and various cell lines (HEK, HeLa, NIH3T3).
Production of transgenic organisms with targeted genomic insertions and dual-labeling of different proteins.
Replacement of problematic antibody-based detection with genetic insertion of multi-epitope 'Kaleidoscope' payloads for enhanced imaging and quantification.
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