The present invention provides novel RNA base editing compositions, systems, methods and uses. Guide RNAs for site-specific RNA editing of RNA encoding transcriptional coactivators YAP1 or TAZ are provided, and compositions and systems comprising the same with a programmable RNA binding protein (e.g. a Cas protein) and/or a base editor. Methods for RNA editing of YAP1 or TAZ are also provided. RNA editing of YAP1 or TAZ is used for targeting phosphorylation sites, and activating transcription of proteins in regenerative therapy for treating cardiac disease.

The present disclosure provides Type VI CRISPR-Cas effector polypeptides that can, when complexed with a guide nucleic acid, modify a target RNA. A Type VI CRISPR-Cas effector polypeptide of the present disclosure can also provide for detection of nucleic acid by cleavage of non-target RNAs. The present disclosure provides methods of modifying a target RNA, and methods of detecting a nucleic acid.

The disclosure includes non-naturally occurring or engineered CRISPR Cas9, each associated with at least one destabilization domain (DD), along with compositions, systems and complexes involving the DD-CRISPR Cas9, nucleic acid molecules and vectors encoding the same, delivery systems involving the same, uses therefor.

Some aspects of this disclosure provide compositions, strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins capable of inducing a cytosine (C) to guanine (G) change in a nucleic acid (e.g., genomic DNA) are provided. In some embodiments, fusion proteins of a nucleic acid programmable DNA binding protein (e.g., Cas9) and nucleic acid editing proteins or protein domains, e.g., deaminase domains, polymerase domains, and/or base excision enzymes are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of a nucleic acid programmable DNA binding protein (e.g., Cas9), and nucleic acid editing proteins or domains, are provided.

The present disclosure provides virus-like particles for delivering gene editing agents such as nucleic acid-programmable DNA-binding proteins (napDNAbps) and base editor fusion proteins (BE-VLPs or eVLPs), and systems comprising such eVLPs. The present disclosure also provides polynucleotides encoding the eVLPs described herein, which may be useful for producing said eVLPs. Also provided herein are methods for editing the genome of a target cell by introducing the presently described eVLPs into the target cell. The present disclosure also provides fusion proteins that make up a component of the eVLPs described herein, as well as polynucleotides, vectors, cells, and kits.

The present invention relates to a novel CRISPR system comprising i) at least one nucleotide sequence encoding at least one Cas13 protein; and ii) at least one gRNA or at least one nucleotide sequence encoding said at least one gRNA capable of hybridizing with one or more viral target RNA molecules, wherein said system comprises a viral 5 UTR or a nucleotide sequence encoding said 5 UTR and/or a viral 3 UTR or a nucleotide sequence encoding said viral 3 UTR, wherein a viral replicase recognition sequence is comprised in at least any one of said 5 UTR or in the nucleotide sequence encoding said 5 UTR, or said 3 UTR or in the nucleotide sequence encoding said 3 UTR, and wherein said system does not comprise a nucleotide sequence encoding a viral replicase and wherein said viral replicase recognition sequence is from the same RNA virus as the one or more viral target RNA molecules. The present invention also relates to a delivery system comprising the novel system and a composition comprising the novel system or the delivery system. The present invention further relates to the medical use of the novel system or in particular to the system for use in a method of preventing or treating a viral disease in a subject. Additionally, the present invention also relates to a kit comprising the novel system and to a method of producing the novel system.

The disclosure provides programmable methylation writers and demethylation erasers for editing the methylation state of RNA targets, e.g., an RNA transcriptome. In particular, the disclosure provides RNA methylation editor polynucleotide contracts and vectors comprising (i) an RNA programmable RNA binding domain (RNApRNAbd); and (ii) an effector domain, wherein the effector domain is capable of adding or removing a methyl group in an RNA. The disclosed RNA methylation editor constructs are capable of achieving limited off-target modifications in RNA molecules. Further, the disclosure provides methods for making and using the programmable methylation editors to modifying the methylation state of RNA. The disclosure further provides complexes comprising a methylation writer protein and a guide RNA molecule and complexes comprising a demethylation eraser protein and a guide RNA molecule. The disclosure further provides pharmaceutical compositions and cells comprising the disclosed fusion proteins and complexes.

The present disclosure provides systems and methods for modifying a target viral nucleic acid in the cytoplasm of a eukaryotic cell.

The disclosure provides programmable methylation writers and demethylation erasers for editing the methylation state of RNA targets, e.g., an RNA transcriptome. In particular, the disclosure provides RNA methylation editor polynucleotide constructs and vectors comprising (i) an RNA programmable RNA binding domain (RNApRNAbd); and (ii) an effector domain, wherein the effector domain is capable of adding or removing a methyl group in an RNA. The disclosed RNA methylation editor constructs are capable of achieving limited off-target modifications in RNA molecules. Further, the disclosure provides methods for making and using the programmable methylation editors to modifying the methylation state of RNA. The disclosure further provides complexes comprising a methylation writer protein and a guide RNA molecule and complexes comprising a demethylation eraser protein and a guide RNA molecule. The disclosure further provides pharmaceutical compositions and cells comprising the disclosed fusion proteins and complexes.