This invention pertains to optimized protein fusion linkers for creating multi-functional chimeric proteins and methods of using the same. Additionally, the invention pertains to chimeric proteins for use in guided endonuclease systems.

The present disclosure provides a Cas9 heterodimer, as well as nucleic acids encoding the Cas9 heterodimer, and host cells comprising the nucleic acids. The present disclosure provides a system that includes a Cas9 heterodimer of the present disclosure and at least one of: a Cas9 guide RNA, and a dimerizing agent. A Cas9 heterodimer of the present disclosure is useful in a wide variety of applications, which are also provided.

Compositions and methods are provided for genome modification of a target sequence in the genome of a plant or plant cell. The methods and compositions employ a guide RNA/Cas endonuclease system to provide an effective system for modifying or altering target sites within the genome of a plant, plant cell or seed. Also provided are compositions and methods employing a guide polynucleotide/Cas endonuclease system for genome modification of a nucleotide sequence in the genome of a cell or organism, for gene editing, and/or for inserting or deleting a polynucleotide of interest into or from the genome of a cell or organism. Once a genomic target site is identified, a variety of methods can be employed to further modify the target sites such that they contain a variety of polynucleotides of interest. Breeding methods and methods for selecting plants utilizing a two component RNA guide and Cas endonuclease system are also disclosed. Compositions and methods are also provided for editing a nucleotide sequence in the genome of a cell.

The present invention provides methods of treating, preventing or delaying the onset of bone marrow failure in Fanconi Anemia patients.

The invention includes materials and methods to generate numerous small RNAs from one polynucleotide construct (synthetic gene) to facilitate RNA-guided multiplex genome editing, modification, inhibition of expression and other RNA-based technologies. The synthetic gene/polynucleotide construct encodes polycistronic RNA components separated by tRNAs, and preferably also includes regulatory components such as a promoter or terminator to form an expression cassette. Once transcribed in a cell, the transcript is processed by the cell to multiple RNA molecules by the endogenous tRNA processing system. The system can be sued for any RNA based gene manipulation method including RNA-mediated genome editing, artificial microRNA mediated gene silencing, small RNA mediated genetic manipulation, double-stranded RNA mediated gene silencing, antisense mechanisms and the like.

Described herein are methods and vectors for rational, multiplexed manipulation of chromosomes within open reading frames (e.g., in protein libraries) or any segment of a chromosome in a cell or population of cells, in which various CRISPR systems are used.

Compositions and methods are provided for the inhibition, treatment and/or prevention of degenerative myelopathy (DM) or amyotrophic lateral sclerosis (ALS).

Provided are RNA polynucleotide sequences referred to as “EXO-Codes.” The RNA polynucleotides comprise one or more nucleotide sequences that facilitate preferential enrichment of secreted membranous vesicles that contain the EXO-Codes. Nucleotide motifs that contribute to these properties of the EXO-Codes are described. Modified eukaryotic cells comprising EXO-Codes are provided, and include lymphocytes such as T cells. EXO-Codes may comprise a cargo that provides a prophylactic or therapeutic effect. Exosome preparations comprising the EXO-codes are provided. Pharmaceutical compositions comprising EXO-Codes, expression vectors encoding them, and methods of using the pharmaceutical formulations are provided. The pharmaceutical compositions may comprise the EXO-Codes within membranous structures, such as exosomes. Cells that express or otherwise include the EXO-Codes are included, as are method for separating membranous structures that contain the EXO-codes from the cells.

Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity and/or improving the specificity of RNA-programmable endonucleases, such as Cas9. For example, provided are guide RNAs (gRNAs) that are engineered to exist in an “on” or “off” state, which control the binding and hence cleavage activity of RNA-programmable endonucleases. Some aspects of this disclosure provide mRNA-sensing gRNAs that modulate the activity of RNA-programmable endonucleases based on the presence or absence of a target mRNA. Some aspects of this disclosure provide gRNAs that modulate the activity of an RNA-programmable endonuclease based on the presence or absence of an extended DNA (xDNA).

A modified U1 snRNA, wherein said modified U1 snRNA has been modified to bind to a nucleotide sequence within the packaging region of a lentiviral vector genome sequence.