Artificial nucleases comprising engineered cleavage half-domains
Inventors
Doyon, Yannick • Miller, Jeffrey C.
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11725218-B2
Publication Date
2023-08-15
Expiration Date
Abstract
Disclosed herein are engineered cleavage half-domains; fusion polypeptides comprising these engineered cleavage half-domains; polynucleotides encoding the engineered cleavage half-domains and fusion proteins; and cells comprising said polynucleotides and/or fusion proteins. Also described are methods of using these polypeptides and polynucleotides, for example for targeted cleavage of a genomic sequence.
Core Innovation
The invention describes artificial nucleases comprising a DNA-binding domain that binds to a target site in a cell's genome and a polypeptide comprising an engineered FokI cleavage half-domain. The cleavage half-domain comprises specified sets of substitution mutations, with residue numbering relative to full-length wild-type FokI. The described variants are designed to favor heterodimerization and suppress homodimerization when used in DNA-binding-domain fusion nucleases.
The addressed problem involves off-target cleavage associated with targeted genomic cleavage in cells. The engineered cleavage half-domains are intended to improve targeted cleavage specificity, with reported examples describing reduced off-target cleavage. In addition, reported figure summaries provide genomic indel measurements for engineered FokI-based nuclease pairs across several target sites, supporting functional nuclease activity in genomic editing assays.
Claims Coverage
The relevant claims identify one independent claim directed to an artificial nuclease, comprising two inventive features.
DNA-binding domain targeting a genomic site
The artificial nuclease comprises a DNA-binding domain that binds to a target site in the genome of a cell.
Engineered FokI cleavage half-domain
The artificial nuclease comprises a polypeptide comprising an engineered FokI cleavage half-domain containing specified substitution mutations, with residue numbering relative to full-length wild-type FokI.
The independent claim covers an artificial nuclease combining a genomic target-binding domain with an engineered FokI cleavage half-domain containing specified substitution mutations.
Stated Advantages
The variants are designed to favor heterodimerization and suppress homodimerization, improving targeted cleavage specificity.
The reported examples describe increased indel activity over earlier heterodimer designs in multiple cell and target contexts.
The reported examples describe reduced off-target cleavage.
The reported examples describe improved donor-directed integration.
Documented Applications
Targeted genomic cleavage and gene modification, including gene-locus inactivation and the creation of insertions or deletions.
Insertion of a transgene into a gene locus and targeted integration.
Homologous recombination and simultaneous editing at multiple loci.
Genomic modification in T-cells, hematopoietic stem cells, and liver cells.
Interested in licensing this patent?