The present invention provides RNA-guided endonucleases, which are engineered for expression in eukaryotic cells or embryos, and methods of using the RNA-guided endonuclease for targeted genome modification in in eukaryotic cells or embryos. Also provided are fusion proteins, wherein each fusion protein comprises a CRISPR/Cas-like protein or fragment thereof and an effector domain. The effector domain can be a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. Also provided are methods for using the fusion proteins to modify a chromosomal sequence or regulate expression of a chromosomal sequence.

Disclosed is a method for editing mitochondrial DNA (mtDNA) within a cell, which include introducing into the cell (a) a DNA cleaving enzyme targeted to the mtDNA sequence to be deleted; (b) a first DNA binding component targeted to a sequence adjacent to the 5′ end of a mtDNA sequence to be deleted; and (c) a second DNA binding component targeted to a sequence adjacent to the 3′ end of the mtDNA sequence to be deleted, where the DNA cleaving enzyme generates a double stranded break (DSB) within the mtDNA sequence to be deleted or generates a single strand nick on the light strand of the mtDNA sequence to be deleted, and wherein the mtDNA sequence between the target sequence for the first DNA binding component and the target sequence for the second DNA binding component is deleted.

The present invention relates in part to nucleic acids encoding proteins, therapeutics comprising nucleic acids encoding proteins, methods for inducing cells to express proteins using nucleic acids, methods, kits and devices for transfecting, gene editing, and reprogramming cells, and cells, organisms, and therapeutics produced using these methods, kits, and devices. Methods and products for altering the DNA sequence of a cell are described, as are methods and products for inducing cells to express proteins using synthetic RNA molecules. Therapeutics comprising nucleic acids encoding gene-editing proteins are also described.

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.

Disclosed are compositions and methods for directing proteins to specific loci in the genome and uses thereof. In one aspect, the disclosed methods allow for directing proteins to specific loci in the genome of an organism, including the steps of providing a fusion protein comprising a DNA localization component and an effector molecule. Preferred embodiments of the disclosure include, but are not limited to, the following fusion proteins: dSaCas9-Clo051, dCas9-Clo051, Xanthomonas-TALE-Clo051, and Ralstonia-TALE-Clo051.

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.

This disclosure relates to Cas9-nucleic acid complexes and uses related thereto. In certain embodiments, the disclosure contemplates transgenic plants and animals genetically engineered to express Cas9-nucleic acid complexes disclosed herein. In certain embodiments, the disclosure relates to methods of treating or preventing, diseases, conditions, cancer, viral infections or other pathogenic infection using vectors configured to express a Cas9-nucleic acid complex disclosed herein.

Disclosed herein are methods and compositions for targeted cleavage of a genomic sequence, targeted alteration of a genomic sequence, and targeted recombination between a genomic region and an exogenous polynucleotide homologous to the genomic region. The compositions include fusion proteins comprising a cleavage domain (or cleavage half-domain) and an engineered zinc finger domain and polynucleotides encoding same. Methods for targeted cleavage include introduction of such fusion proteins, or polynucleotides encoding same, into a cell. Methods for targeted recombination additionally include introduction of an exogenous polynucleotide homologous to a genomic region into cells comprising the disclosed fusion proteins.

Engineered CRISPR from Prevotella and Francisella 1 (Cpf1) nucleases with altered and improved target specificity and their use in genomic engineering, epigenomic engineering, genome targeting, genome editing, and in vitro diagnostics.

The invention provides for systems, methods, and compositions for altering expression of target gene sequences and related gene products. Provided are structural information on the Cas protein of the CRISPR-Cas system, use of this information in generating modified components of the CRISPR complex, vectors and vector systems which encode one or more components or modified components of a CRISPR complex, as well as methods for the design and use of such vectors and components. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for utilizing the CRISPR-Cas system. In particular the present invention comprehends optimized functional CRISPR-Cas enzyme systems, wherein the guide sequence is modified by secondary structure to increase the specificity of the CRISPR-Cas system and whereby the secondary structure can protect against exonuclease activity and allow for 5′ additions to the guide sequence.