The present invention is related to the field of gene editing. In particular, the gene editing is directed toward single nucleotide base editing. For example, such single nucleotide base editing results in a conversion of a OG base pair to a T*A base pair. The high accuracy and precision of the presently disclosed single nucleotide base gene editor is accomplished by an NmeCas9 nuclease that is fused to a nucleotide deaminase protein. The compact nature of the NmeCas9 coupled with a larger number of compatible protospacer adjacent motifs provide the Cas9 fusion constructs contemplated herein to have a gene editing window that can edit sites that are not targetable by other conventional SpyCas9 base editor platforms.

The present invention belongs to the field of plant genetic engineering. Specifically, the invention relates to a method for base editing in plants. More particularly, the invention relates to a method for performing efficient base editing to a target sequence in the genome of a plant (such as a crop plant) by a Cas9-cytidine deaminase fusion protein, as well as plants produced through said method and progenies thereof.

Engineered bifunctional adenine and cytosine base editor (BACE) variants that enable expanded amino acid modifications and methods of using the same. Described herein are fusion proteins containing adenosine deaminases, cytidine deaminases, catalytically impaired CRISPR-Cas proteins (e.g., Cas9, CasX or Cas12 nucleases), linkers, nuclear localization signals (NLSs) and uracil-n-glycosylase inhibitors (UGIs) that enable the CRISPR-guided programmable introduction of simultaneous A-to-G (T-to-C) and C-to-T (G-to-A) substitutions in DNA.

The present invention discloses a system for targeted gene editing and related uses. Also disclosed are related cells.

Examples provided herein are related to detecting methylcytosine and its derivatives using S-adenosyl-L-methionine analogs (xSAMs). Compositions and methods for performing such detection are disclosed. A target polynucleotide may include cytosine (C) and methylcytosine (mC). The method may include (a) protecting the C in the target polynucleotide from deamination; and (b) after step (a), deaminating the mC in the target polynucleotide to form thymine (T). Protecting the C from deamination may include adding a protective group to the 5 position of the C, e.g., using a methyltransferase enzyme that adds the first protective group from an xSAM.

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 proteins or protein 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 proteins or domains, are provided.

The present invention features compositions comprising fusion polypeptides comprising an RNA binding polypeptide operationally linked to an RNA modifying enzyme (e.g., adenosine deaminase, cytidine deaminase), and methods of use therefore.

The present invention provides for methods and compositions for specifically targeting a nuclei type in a multinucleated cell or population of multinucleated cells, wherein the composition comprises one or more nucleotide sequences operably linked to one or more regulatory sequences specifically active in the nuclei.

Provided herein are methods of delivering “split” Cas9 protein or nucleobase editors into a cell, e.g., via a recombinant adeno-associated vims (rAAV), to form a complete and functional Cas9 protein or nucleobase editor. The Cas9 protein or the nucleobase editor is split into two sections, each fused with one part of an intein system (e.g., intein-N and intein-C encoded by the dnaE-n and dnaE-c genes, respectively). Upon co-expression, the two sections of the Cas9 protein or nucleobase editor are ligated together via intein-mediated protein splicing. Nucleic acid molecules encoding the N-terminal portion of a Cas9 protein or a nucleobase editor fused to an intein, and nucleic acid molecules encoding the C-terminal portion of a Cas9 protein or nucleobase editor, are provided. Recombinant AAV vectors (e.g, vectors comprising one or more of these nucleic acid molecules each comprising an intein) and particles for the delivery of the split Cas9 protein or nucleobase editor, compositions comprising such AAV vectors and particles, and methods of using such rAAV vectors and particles are also provided. Methods of administering such compositions and AAV particles to a subject are further provided. Cells and compositions comprising these nucleic acid molecules rAAV vectors, and rAAV particles are also provided.

Provided are fusion proteins and related molecules useful for conducting base editing with reduced or no off-target mutations. The fusion proteins may include a first fragment comprising a nucleobase deaminase or a catalytic domain thereof, a second fragment comprising a nucleobase deaminase inhibitory domain, and a protease cleavage site between the first fragment and the second fragment. Also provided are improved prime editing systems, including prime editing guide RNA with improved stability.