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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. In this case, a single fusion protein is expressed in a cell and, without wishing to be bound by theory, is believed to cleave DNA as a result of formation of an intramolecular dimer of the cleavage half-domains. Only essential between the notes of the musician and, in fact, the program writes the notes on a score. A target site generally has a length of at least 9 nucleotides and, accordingly, is bound by a zinc finger binding domain comprising at least three zinc fingers. Patent Publications 2007/0134796 and, herein incorporated by reference in their entireties. In some case, the cell does not naturally produce Cas protein and is genetically engineered to produce a Cas protein.



In certain embodiments, the ZFN or TALEN comprise a cleavage half-domain from the FokI restriction enzyme, and two such fusion proteins are expressed in a cell. These polynucleotides can be inserted into a vector and the vector can be introduced into a cell (see below for additional disclosure regarding vectors and methods for introducing polynucleotides into cells). In certain embodiments, free private sex cam polynucleotides encoding the fusion proteins are constructed. Methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art. For example, methods for the design and construction of fusion proteins comprising DNA-binding domains (e.g., zinc finger domains, TALEs) and regulatory or cleavage domains (or cleavage half-domains), and polynucleotides encoding such fusion proteins, are described in U.S. In these embodiments, dimerization of the cleavage half-domains to form a functional nuclease is brought about by binding of the fusion proteins to sites on opposite DNA strands, with the 3' ends of the binding sites being proximal to each other. Engineered cleavage half-domains described herein can be prepared using any suitable method, for example, by site-directed mutagenesis of wild-type cleavage half-domains (Fok I) as described in U.S. Selection of a target site in a genomic region of interest in cellular chromatin of any gene for binding by a DNA-binding domain (e.g., a target site) can be accomplished, for example, according to the methods disclosed in U.S.



Pat. Nos. 6,453,242 and 6,794,136), one or more of the individual zinc fingers of a multi-finger binding domain can bind to overlapping quadruplet subsites. Soldiering on through a pandemic is stressful, so it’s all the more important that we take advantage of what small human joys remain right now. The pandemic has a profound effect on all five senses. The CRISPR (clustered regularly interspaced short palindromic repeats) locus, which encodes RNA components of the system, and the cas (CRISPR-associated) locus, which encodes proteins (Jansen et al., 2002. Mol. In these embodiments, both fusion proteins bind to the same DNA strand, with the binding site of the first fusion protein containing the zinc finger domain nearest the carboxy terminus located to the 5' side of the binding site of the second fusion protein containing the zinc finger domain nearest the amino terminus. The disclosed methods and compositions include fusion proteins comprising a DNA-binding domain (e.g., ZFP, TALE, etc.) and a regulatory domain or cleavage (e.g., nuclease) domain (or a cleavage half-domain), in which the DNA-binding domain, by binding to a sequence in cellular chromatin in one or more plant genes, induces cleavage and targeted integration of one or more exogenous sequences (including transgenes) into the vicinity of the target sequence.



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 still further embodiments, the nuclease comprises a compact TALEN (cTALEN). In certain embodiments, the nuclease comprises a CRISPR/Cas system. Nucleases can be screened for activity prior to use, for example in a yeast-based chromosomal system as described in U.S. Activity of the CRISPR/Cas system comprises of three steps: (i) insertion of foreign DNA sequences into the CRISPR array to prevent future attacks, in a process called `adaptation`, (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference with the foreign nucleic acid. The meganuclease cleavage domain is active as a monomer and does not require dimerization for activity. In certain embodiments of the disclosed fusion proteins, the amino acid sequence between the zinc finger domain and the cleavage domain (or cleavage half-domain) is denoted the "ZC linker." The ZC linker is to be distinguished from the inter-finger linkers discussed above. Finally, Cas9 mediates cleavage of target DNA to create a double-stranded break within the protospacer.