Building IP: Sangamo Life Technologies Corp. Patent Application Re "DELIVERY OF TARGET SPECIFIC NUCL
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Mutations include substitutions (of a wild-type amino acid residue for a different residue, insertions (of one or more amino acid residues) and/or deletions (of one or more amino acid residues). Alternatively, nucleases may be assembled in vivo at the nucleic acid target site using so-called "split-enzyme" technology (see e.g. U.S. As such, gene modification can be achieved using a nuclease, for example an engineered nuclease. Thus, in the methods and compositions described herein, it is understood that the term "Cas" includes all Cas variant proteins, both natural and engineered. In any of the nucleases described herein, the nuclease can comprise an engineered TALE DNA-binding domain and a nuclease domain (e.g., endonuclease and/or meganuclease domain), also referred to as TALENs. In certain embodiments, the nuclease comprises a catalytically inactive cleavage domain (e.g., FokI and/or Cas protein). In certain embodiments, the cleavage domain comprises a FokI cleavage domain used to generate the crystal structures 1FOK.pdb and 2FOK.pdb (see Wah et al.
In certain embodiments, the nuclease comprises a CRISPR/Cas system. In other embodiments, the TALE-nuclease is a mega TAL. In certain embodiments, described herein are artificial nucleases comprising a DNA-binding binding domain and cleavage (nuclease) domain. 577-582. The catalytically inactive cleavage domain may, in combination with a catalytically active domain act as a nickase to make a single-stranded cut. Cas protein, which includes Cas protein or a fragment thereof, as well as derivatives of Cas protein or a fragment thereof, may be obtainable from a cell or synthesized chemically or by a combination of these two procedures. Cell 99:443-446; Knoepfler et al. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. Examples of this type of "pioneer" DNA binding domain are found in certain steroid receptor and in hepatocyte nuclear factor 3 (HNF3) (Cordingley et al. Fusion molecules comprise a DNA-binding domain and a functional domain (e.g., a transcriptional activation or repression domain).
Additional exemplary activation domains include, Oct 1, Oct-2A, Sp1, AP-2, and CTF1 (Seipel et al. 9:499-504. Additional exemplary activation domains include, but are not limited to, OsGAI, HALF-1, C1, AP1, ARF-5, -6, -7, and -8, CPRF1, CPRF4, MYC-RP/GP, and TRAB1. Hence, the functional component can include, but is not limited to, various transcription factor domains, such as activators, repressors, co-activators, co-repressors, and silencers. Engineering methods include, but are not limited to, rational design and various types of selection. Methods and compositions for engineering these TALEN proteins for robust, site specific interaction with the target sequence of the user's choosing have been published (see U.S. In other embodiments, the nuclease is a zinc finger nuclease (ZFN) or TALE DNA binding domain-nuclease fusion (TALEN). In certain embodiments, Cas protein may be a "functional derivative" of a naturally occurring Cas protein. In some embodiments, other Cas proteins may be used. These are single chain fusion proteins linking a TALE DNA binding domain to a TevI nuclease domain. The functional component/domain of a fusion molecule can be selected from any of a variety of different components capable of influencing transcription of a gene once the fusion molecule binds to a target sequence via its DNA binding domain.
