The present invention features compositions and methods for editing a gene associated with Shwachman Diamond Syndrome (SDS) using a programmable nucleobase editor, such that the gene is permissive for transcription and generates a functional gene product (e.g., providing a splice site and/or altering a nonsense mutation).

The disclosure provides for systems, methods, and compositions for targeting and editing nucleic acids. In particular, the invention provides non-naturally occurring or engineered RNA-targeting systems comprising a RNA-targeting Cas13 protein, at least one guide molecule, and at least one adenosine deaminase protein or catalytic domain thereof.

The present disclosure provides a fusion polypeptide comprising: a) an enzymatically active RNA-guided endonuclease that introduces a single-stranded break in a target DNA; and b) an error-prone DNA polymerase. The present disclosure provides a system comprising: a) a fusion polypeptide of the present disclosure; and b) a guide RNA. The present disclosure provides a cell comprising a fusion polypeptide of the present disclosure, or a system of the present disclosure. The present disclosure provides a method of mutagenizing a target polynucleotide.

Provided are methods for using chimeric proteins to produce RNA modifications that can be detected by sequencing methods, including methods detecting relative translation rates of various mRNAs. Also provided herein are compositions comprising chimeric proteins, wherein the chimeric proteins comprise a RNA editing protein and a ribosomal protein.

The disclosure provides for systems, methods, and compositions for targeting and editing nucleic acids. In particular, the invention provides non-naturally occurring or engineered RNA-targeting systems comprising a RNA-targeting Cas13 protein, at least one guide molecule, and at least one adenosine deaminase protein or catalytic domain thereof.

Some aspects of this disclosure provide 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 of Cas9 and nucleic acid editing enzymes or enzyme domains, e.g., deaminase domains, 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 Cas9 and nucleic acid editing enzymes or domains, are provided.

Provided herein are compositions and methods for detection of N.sup.6-methyladenosine (m.sup.6A) in ribonucleic acid (RNA). The provided compositions include fusion proteins that can be used to edit RNA and detect m6A residues. Also provided are nucleic acids, vectors, constructs, host cells, and transgenic animals that encode or express such fusions proteins.

Provided herein are compositions that include proteins that include a mitochondrial localization amino acid sequence attached to a base editor fusion protein including a nucleotide base-modifying enzyme and an RNA-guided DNA endonuclease enzyme with modified endonuclease activity. Provided herein are nucleic acids encoding the proteins and vectors including the nucleic acids. Provided herein are pharmaceutical compositions including the compositions, proteins, nucleic acid and vectors. Provided herein are related methods for modifying mitochondrial DNA and treating mitochondrial disorders.

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for engineering Cas9 and Cas9 variants that have increased activity on target sequences that do not contain the canonical PAM sequence (e.g., NGG). In some embodiments, fusion proteins comprising such Cas9 variants and nucleic acid editing domains, e.g., deaminase domains, are provided.

The invention provides a method for enhanced genome engineering using CRISPR/Cas system tethered to exonucleases via different systems. By connecting Cas9 and exonucleases via heterodimeric peptides that form coiled-coils or via heterodimerization systems, greater indel mutations at desired genomic region occurs, resulting in higher genome editing rate. This novel improved CRISPR/Cas system can be exploited in different cell origins and organisms in various fields, where genome engineering is required and desired.