Compositions for linking DNA-binding domains and cleavage domains

Inventors

Miller, Jeffrey C. • Paschon, David • Rebar, Edward J.

Assignees

Sangamo Therapeutics Inc

Abstract

Disclosed herein are compositions for linking DNA binding domains and cleavage domains (or cleavage half-domains) to form non-naturally occurring nucleases with alternative configurations. Also described are methods of making and using compositions comprising these linkers.

Core Innovation

The invention relates to dimeric fusion molecules in which a first fusion molecule and a second fusion molecule each comprise a DNA binding domain and a cleavage domain. The cleavage domains are configured to dimerize when the DNA binding domains are bound to two target sites that are separated by 7 base pairs, 8 base pairs, or 9 base pairs.

Each fusion molecule further includes an amino acid linker between the DNA binding domain and the cleavage domain. The linker is selected from groups of specific amino acid linker sequences, and the fusion molecule architecture enables non-naturally occurring nuclease configurations, including head-to-tail and head-to-head arrangements, and expanded gap spacings beyond conventional separations.

The disclosed systems are used for targeted DNA cleavage and chromatin editing, including donor-mediated sequence insertion/correction mechanisms. The document further describes portability across dimer architectures and activity assessments at target loci and chromatin loci, including engineered cell lines, and comparisons to conventional tail-to-tail configurations.

Claims Coverage

The independent claims define dimeric fusion architectures in which two cleavage domains dimerize when their respective DNA-binding domains bind to two target sites separated by a specified number of base pairs, with each first fusion molecule including a specified amino acid linker. Across the independent claims, the main inventive features are dimerization-activated nuclease coupling, specified target-site spacing (7, 8, or 9 base pairs), and specific linker selections between DNA-binding domains and cleavage domains.

Across the independent claims, coverage centers on dimeric fusion molecules that activate cleavage-domain dimerization through DNA-binding-domain occupancy of two target sites with specified base-pair separation (7, 8, or 9 base pairs), together with the requirement that the first fusion molecule includes an amino acid linker between the DNA-binding domain and the cleavage domain selected from specified SEQ ID NO linker sequences.

Stated Advantages

Documented Applications