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Exemplary selection methods, including phage display and two-hybrid systems, are disclosed in U.S. See, for example, Remington’s Pharmaceutical Sciences, 17th ed., 1985; and U.S. See, for example, Roberts et al. See, for example, Ogawa et al. Suitable domains for achieving activation include the HSV VP16 activation domain (see, e.g., Hagmann et al. See, for example, Chem et al. WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB 2,338,237. In addition, enhancement of binding specificity for zinc finger binding domains has been described, for example, in U.S. Specificity of these TAL effectors depends on the sequences found in the tandem repeats. Since the specificity of TtAgo cleavage is directed by the guide DNA, a TtAgo-DNA complex formed with an exogenous, investigator-specified guide DNA will therefore direct TtAgo target DNA cleavage to a complementary investigator-specified target DNA. For cleavage of mammalian genomic DNA, it would be preferable to use of a version of TtAgo codon optimized for expression in mammalian cells.
For your privacy, make sure you always use one, especially while banking or viewing a site that you might not want people to know you’re visiting. Use of the TtAgo-guide DNA system (or orthologous Ago-guide DNA systems from other organisms) allows for targeted cleavage of genomic DNA within cells. Parameters for targeted cleavage and targeted sequence alteration using zinc finger-FokI fusions are provided elsewhere in this disclosure. In this way, one may create a targeted double-strand break in DNA. Alternatively, nucleases may be assembled in vivo at the nucleic acid target site using so-called «split-enzyme» technology (see e.g. U.S. Alternatively, a single protein comprising two cleavage half-domains can be used. LNPs as describe herein can include fusion molecules comprising DNA-binding domains (e.g., ZFPs or TALEs, CRISPR/Cas components such as single guide RNAs) and a heterologous regulatory (functional) domain (or functional fragment thereof) are also provided. 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.
Nature Genet. 25:338-342. Additional exemplary repression domains include, but are not limited to, ROM2 and AtHD2A. Nature 441:656-659; Paques et al. A «tautomer» refers to a proton shift from one atom of a molecule to another atom of the same molecule. The present invention contemplates various stereoisomers and mixtures thereof and includes «enantiomers», which refers to two stereoisomers whose molecules are nonsuperimposeable mirror images of one another. In general, two fusion molecules are required for cleavage if the fusion molecules comprise cleavage half-domains. The two cleavage half-domains can be derived from the same endonuclease (or functional fragments thereof), or each cleavage half-domain can be derived from a different endonuclease (or functional fragments thereof). In some embodiments, the TALEN comprises an endonuclease (e.g., FokI) cleavage domain or cleavage half-domain. In certain embodiments, the nuclease comprises a meganuclease (homing endonuclease) or a portion thereof that exhibits cleavage activity. Similarly, a cleavage half-domain can be derived from any nuclease or portion thereof, as set forth above, that requires dimerization for cleavage activity. In still further embodiments, the nuclease comprises a compact TALEN (cTALEN).
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