Systems and methods for targeted gene modification, targeted insertion, perturbation of gene transcripts, and nucleic acid editing. Novel nucleic acid targeting systems comprise components of CRISPR systems and non-LTR retrotransposon elements.

Disclosed herein are polypeptides, polynucleotides encoding, cells and organisms comprising novel DNA-binding domains, including TALE DNA-binding domains. Also disclosed are methods of using these novel DNA-binding domains for modulation of gene expression and/or genomic editing of endogenous cellular sequences.

The invention provides highly effective and versatile CRISPR/Cas protein variants, compositions, methods and uses thereof in gene editing. More specifically, the invention relates to PAM-reduced or PAM-abolished Cas proteins and chimeras, complexes and conjugates thereof, genetic editing systems and to therapeutic and non-therapeutic methods and uses of the PAM-reduced or PAM-abolished Cas proteins.

The present invention relates to a method for in vitro transcription of a linear template DNA which is produced using an immobilized restriction endonuclease. The invention also relates to mutated restriction enzymes which are suitable for immobilization and a solid support to which these restriction enzymes are immobilized. Further, the present invention relates to an enzyme reactor containing said immobilized restriction endonuclease which enzyme reactor can be used for preparing linearized template DNA. Finally, the present invention relates to the use of said enzyme reactor for preparing a linear template DNA for in vitro transcription. In addition, the present invention relates to a kit comprising the immobilized restriction endonuclease.

Disclosed herein are polypeptides, polynucleotides encoding, cells and organisms comprising novel DNA-binding domains, including TALE DNA-binding domains. Also disclosed are methods of using these novel DNA-binding domains for modulation of gene expression and/or genomic editing of endogenous cellular sequences.

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.

Methods for increasing specificity of RNA-guided genome editing, e.g., editing using CRISPR/Cas9 systems, using truncated guide RNAs (tru-gRNAs).

Disclosed herein are methods and compositions for the repair of site specific nuclease binding sites by targeted integration and/or targeted excision of one or more sequences into a cell.

Disclosed herein are polypeptides, polynucleotides encoding, cells and organisms comprising novel DNA-binding domains, including TALE DNA-binding domains. Also disclosed are methods of using these novel DNA-binding domains for modulation of gene expression and/or genomic editing of endogenous cellular sequences.

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.