Disclosed herein are methods and compositions for targeted integration of an exogenous sequence into the human PPP1R12C locus, for example, for expression of a polypeptide of interest.

The presently claimed invention offers programmable and precise regulation of Cas9 functions by utilizing a set of compact Cas9 derivatives created by deleting conserved HNH and/or REC-C domains based on the structural information across variant class 2 CRISPR effectors. In addition, a novel strategy for engineering the dimeric gRNA-guided nuclease by splitting the mini-dSaCas9 and fusing the FokI domain right after the split point is claimed to increase the on-target DNA cleavage efficiency and potentially reduce the off-target effect because of a closer proximity of dimeric Fold nuclease to the target sequence. By combining the optimized and compact gRNA expression cassette and the downsized SaCas9 derivatives, the entire CRISPR/Cas system with different effector domains for transactivation, DNA cleavage and base editing is loaded into a single AAV virus. Such an all-in-one AAV-CRISPR/Cas9 system will be particularly appealing in biomedical applications that require safe and efficient delivery in vivo.

The invention provides for systems, methods, and compositions for targeting RNA. In particular, the invention provides a non-naturally occurring or engineered RNA-targeting system comprising an RNA-targeting Cas protein and at least one RNA-targeting guide RNA, wherein said RNA-targeting guide RNA is capable of hybridizing with a target RNA in a cell.

Provided herein are compositions aid methods for barcoding mammalian cells. The compositions and methods provided herein further provide methods for tracing such barcoded cells ex vivo or in vivo during the life time of an organism. In one aspect, a method of forming a barcoded cell is provided. The method includes expressing in a cell a heterologous cleaving protein complex including a sequence-specific DNA-binding domain and a nucleic acid cleaving domain. The sequence-specific DNA-binding domain targets the nucleic acid cleaving domain to a genomic nucleic acid sequence, thereby forming a genomic nucleic acid sequence bound to the heterologous cleaving protein complex.

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.

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.

Methods and compositions for modifying T-cells in which PD1 and/or CTLA-4 is repressed and/or inactivated using fusion proteins such as artificial transcription factors and nucleases.

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.

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.

Materials and Methods related to gene targeting (e.g., gene targeting with transcription activator-like effector nucleases; “TALENS”) are provided.