CRISPR RNA-guided nucleases are routinely used for sequence-specific manipulation of DNA. While CRISPR-based DNA editing has become routine, analogous methods for editing RNA have yet to be established. Here we repurpose the type III-A CRISPR RNA-guided nuclease for sequence-specific cleavage of the SARS-CoV-2 genome. The type III cleavage reaction is performed in vitro using purified viral RNA, resulting in sequence-specific excision of 6, 12, 18 or 24 nucleotides. Ligation of the cleavage products is facilitated by a DNA splint that bridges the excision and RNA ligase is used to link the RNA products before transfection into mammalian cells. The SARS-CoV-2 RNA is infectious and standard plaque assays are used to recover viral clones. Collectively, this work demonstrates how type III CRISPR systems can be repurposed for sequence-specific editing of RNA viruses including SARS-CoV-2 and more generally for gene therapy.

The present disclosure provides engineered Class 1 Type I CRISPR-Cas (Cascade) systems that comprise multi-protein effector complexes, nucleoprotein complexes comprising Type I CRISPR-Cas subunit proteins and nucleic acid guides, polynucleotides encoding Type I CRISPR-Cas subunit proteins, and guide polynucleotides. Also, disclosed are methods for making and using the engineered Class 1 Type I CRISPR-Cas systems of the present invention.

Phenotypically modified bioengineered microbial spores are provided. The microbial spores may be used in various spore-based technologies such as probiotics, biomaterials and vaccines.

Engineered CRISPR-Cas9 nucleases with altered and improved PAM specificities and their use in genomic engineering, epigenomic engineering, and genome targeting.

Circular nucleic acid vectors that provide for persistently high levels of protein expression are provided. The circular vectors of the subject invention are characterized by being devoid of expression-silencing bacterial sequences, where in many embodiments the subject vectors include a unidirectional site-specific recombination product hybrid sequence in addition to an expression cassette. Also provided are methods of using the subject vectors for introduction of a nucleic acid, e.g., an expression cassette, into a target cell, as well as preparations for use in practicing such methods. The subject methods and compositions find use in a variety of different applications, including both research and therapeutic applications. Also provided is a highly efficient and readily scalable method for producing the vectors employed in the subject methods, as well as reagents and kits/systems for practicing the same.

The present disclosure provides methods and systems for identification of genomic regions for therapeutic targeting. A method for identifying one or more genomic regions for therapeutic targeting, which may facilitate re-programming of a cell from one phenotypic state to another, may comprise: providing single-cell RNA-seq data for a plurality of diseased cells and a plurality of normal cells of a cell type; mapping the single-cell RNA-seq data for the plurality of diseased cells and the plurality of normal cells into a latent space corresponding to a plurality of phenotypic states of the cell type; identifying, based at least in part on a topology of the latent space, the one or more genomic regions for therapeutic targeting; and electronically outputting the one or more genomic regions for therapeutic targeting.

Compositions and methods are provided for the inhibition, treatment and/or prevention of Huntington's disease and related disorders.

The present application provides materials and methods for treating a patient with a titin-based myopathy, particularly a titin-based cardiomyopathy, and/or other titinopathy. In addition, the present application provides materials and methods for editing the titin gene in a cell by genome editing.

The invention relates to methods, kits, and compositions for reducing the level of or eliminating a nucleic acid vector in situ. The invention encompasses compositions and methods for selectively eradicating nucleic acid vectors in the microbiota using packaged phagemids. The microbiota can be intestinal and the packaged phagemids can be administered orally. The phagemid encodes a nuclease or other enzyme that genetically modifies the nucleic acid vector so that the nucleic acid vector can be inactivated or eliminated.

In alternative embodiments, provided are compositions, including recombinant expression systems and vectors, products of manufacture and kits, and methods, for remotely-controlled and non-invasive manipulation of intracellular nucleic acid expression, genetic processes, function and activity in live cells such as T cells in vivo, for example, activating, adding functions or changing or adding specificities for immune cells, for monitoring physiologic processes, for the correction of pathological processes and for the control of therapeutic outcomes. In alternative embodiments, provided are blue-light-mediated light-inducible nuclear translocation and dimerization (LINTAD) systems for gene regulation to control cell activation based on the integration of light-sensitive LOV2-based nuclear localization, light-induced active transportation via the biLINuS motif, and CRY2-CIB1 dimerization that feature high spatiotemporal control to control or alter cell activities in vivo, for example, to limit CAR T cell activity to the tumor site for immunotherapy applications.