Described herein are methods and compositions for genomic editing. Endonucleases for genomic editing involve inducing breaks in double stranded DNA, for which knock-ins are notoriously inefficient for relying on random integration of homologous DNA sequences into the break site by repair proteins. To address these issues, described herein are novel recombinant fusion proteins that actively recruit linear DNA inserts in closer proximity to the genomic cleavage site, increasing integration efficiency of large DNA fragments into the genome. Such improvements to genomic editing technology allow one to use lower linear DNA concentrations without sacrificing efficiency and can be further combined with other features, such as fluorescent protein reporting systems.

The present invention relates to methods and compositions for modifying a target site in the genome of a cell. Fusion proteins including one or more DNA binding domains and one or more heterologous domains, such as DNA modifying domains, connected by improved linker sequences are provided. Codon optimized polynucleotides encoding fusion proteins including one or more DNA binding domains and one or more heterologous domains connected by improved linker sequences are provided.

Described herein is a chimeric antigen receptor (CAR) platform with the ability to (a) serve as an ON/OFF switch (with the ability for tenability/titrability), (b) sense multiple antigens and perform logic computations, and/or (c) independently regulate multiple signaling pathways. The compositions provided herein permit the degree of control and discrimination necessary to optimize CAR T cell therapy. Also described herein are cells comprising such compositions and the use of these compositions and/or cells in the treatment of cancer.

The disclosure provides systems, methods, and compositions for a target specific nuclease and a blunting enzyme to correct frameshift mutations for genome editing and treatment of diseases. In some embodiments, the target specific nuclease and the blunting enzyme are combined with a guide RNA and/or a microhomology-mediated end joining (MMEJ) inhibitor.

The invention relates to modified helicases with reduced unbinding from polynucleotides. The helicases can be used to control the movement of polynucleotides and are particularly useful for sequencing polynucleotides.

To address the limitations deriving from the unspecific genomic cleavages of the Streptococcus pyogenes Cas9 (SpCas9) and to identify variants with higher cleavage fidelity, the present invention describes a yeast-based assay which allows to simultaneously evaluate the on- and off-target activity towards two engineered genomic targets. The screening of SpCas9 variants obtained by random mutagenesis of the Red-II domain allowed the identification of hits with increased on/off ratios. The best performing nuclease, evoCas9, was isolated through the combination of the identified mutations within a single variant. Side by side analyses with previously reported rationally designed variants demonstrated a significant improvement in fidelity of evoCas9 of the present invention.

A method for re-expression of hypermethylated RASAL1, hypermethylated LRFN2, and hypermethylated KLOTHO based on an inactivated CRISPR-based system and a DNA dioxygenase as well as a gRNA guiding the construct to the RASAL1, LRFN2, and KLOTHO gene for demethylation of hypermethylated RASAL1, hypermethylated LRFN2, and hypermethylated KLOTHO, in particular, hypermethylated RASAL1, LRFN2, and KLOTHO promoter, thus, allowing re-expression of RASAL1, LRFN2, and KLOTHO for the treatment of fibrosis, cancer or neuronal disorders in a subject is provided. A kit of parts for allowing re-expression of hypermethylated RASAL1, hypermethylated LRFN2, and hypermethylated KLOTHO in a subject, a vector or vector system, and nucleic acid constructs are also provided.

The invention provides an improved genome editing construct which is capable of editing a target sequence in an HDR-dependent manner (i.e., “HDR-dependent genome editors”) with increased efficiency and reduced indel formation and which does not require a dividing cell. In particular, the instant specification provides a new fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) (e.g., Cas9) with a nickase activity and a single-stranded DNA binding protein (e.g., Rad51) which edits a target DNA in an HDR-dependent manner with greater efficiency (e.g., increased rate of induced HDR) and/or with a lower rate or occurrence of indel formation.

The disclosure provides compositions and methods that make use of a target protein that is capable of binding to a small molecule in order to form a complex, and a binding member that specifically binds to the complex, wherein the target protein is derived from a non-human protein and the small molecule is an inhibitor of the non-human protein. The non-human protein may be derived from a viral, bacterial, fungal or protozoal protein. These compositions and methods permit the controlled interaction of polypeptides that are individually fused to the target protein and binding member, respectively, and can be used to control the activity of dimerization-inducible proteins such as split transcription factors and split chimeric antigen receptors through the addition of the small molecule. The disclosure provides expression vectors, binding members, dimerization-inducible proteins, nucleic acids, cells, viral particles, kits, systems and methods that involve these components.

Some aspects of this disclosure provide compositions, methods, and kits for improving the specificity of RNA-programmable endonucleases, such as Cas9. Also provided are variants of Cas9, e.g., Cas9 dimers and fusion proteins, engineered to have improved specificity for cleaving nucleic acid targets. Also provided are compositions, methods, and kits for site-specific nucleic acid modification using Cas9 fusion proteins (e.g., nuclease-inactivated Cas9 fused to a nuclease catalytic domain or a recombinase catalytic domain). Such Cas9 variants are useful in clinical and research settings involving site-specific modification of DNA, for example, genomic modifications.