The present disclosure is concerned with proteins, methods, compositions, and kits for mapping of methylation status of nucleic acids, including 5-methylcytosine and 5-hydroxymethyl cytosine (5 hmC). In one embodiment, proteins are provided that selectively act on certain modified cytosines of target nucleic acids and converts them to thymidine or modified thymidine analogues. In another embodiment, proteins are provided that selectively act on certain modified cytosines of target nucleic acids and converts them to uracil or thymidine and selectively do not act on other certain modified cytosines of target nucleic acids. Also provided are compositions and kits that include one or more of the proteins and methods for using one or more of the proteins.

It is an object of the present invention to provide a method for editing or modifying plant genomes (a nuclear genome, a plastid genome, and a mitochondrial genome), and in particular, the editing or modification of a single nucleotide. Specifically, the present invention relates to a method for editing genomic DNAs in plant cells, namely, a nuclear genomic DNA, a plastid genomic DNA and a mitochondrial genomic DNA, wherein the method comprises converting target nucleotides on these genomic DNAs to other nucleotides. This conversion is carried out, for example, with cytidine deaminase, and in particular, with the aforementioned enzyme using a double-stranded DNA as a substrate.

Methods and compositions are provided for identifying any of the presence, location and phasing of methylated and/or hydroxymethylated cytosines in nucleic acids including long stretches of DNA. In some embodiments, the method may comprise reacting a first portion (aliquot) of a nucleic acid sample with a dioxygenase and optionally a glucosyltransferase in a reaction mixture containing the nucleic acid followed by a reaction with a cytidine deaminase to detect and optionally map .sup.5mC in a DNA. Optionally, a second portion can be reacted with glucosyltransferase followed by reaction with a cytidine deaminase to detect and optionally map .sup.5hmC in a DNA.

The present invention provides a method of modifying a targeted site of a double stranded DNA of a monocot cell, comprising a step of contacting a complex wherein a nucleic acid sequence-recognizing module that specifically binds to a target nucleotide sequence in the given double stranded DNA and a nucleic acid base converting enzyme are bonded, with said double stranded DNA, to convert one or more nucleotides in the targeted site to other one or more nucleotides or delete one or more nucleotides, or insert one or more nucleotides into said targeted site, without cleaving at least one strand of said double stranded DNA in the targeted site, wherein the double stranded DNA is contacted with the complex by introducing a nucleic acid encoding the complex into the monocot cell. Furthermore, also provided is a complex used for the method, wherein a nucleic acid sequence-recognizing module that specifically binds to a target nucleotide sequence in a double stranded DNA of a monocot cell and a nucleic acid base converting enzyme are bonded.

Compositions and methods comprising uracil stabilizing polypeptides for targeted editing of nucleic acids are provided. Compositions comprise uracil stabilizing polypeptides. Also provided are fusion proteins comprising i) a DNA-binding polypeptide; ii) a deaminase; and iii) a uracil stabilizing polypeptide (USP). The fusion proteins include RNA-guided nucleases fused to deaminases and further fused to a USP, optionally in complex with guide RNAs. Compositions also include nucleic acid molecules encoding the USPs or the fusion proteins. Vectors and host cells comprising the nucleic acid molecules encoding the USPs or the fusion proteins are also provided.

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. In some embodiments, fusion proteins comprising such Cas9 variants and nucleic acid editing domains, e.g., deaminase domains, are provided.

The present disclosure is generally directed to gene editing in plant chloroplast and plant mitochondrial double-stranded DNA. Disclosed herein are cytosine base editors tailored for chloroplast and mitochondrial genomes in plants using plant-specific chloroplast and mitochondrial targeting peptides, a TALE, and a DNA deaminase. The systems of the present disclosure include DNA vectors and protocols to use them for gene editing in plants.

Disclosed in the present specification are: dead Cas12f1 having the nucleic acid cleavage activity removed; and a fusion protein in which a functional domain is fused to the dead Cas12f1. The dead Cas12f1 and the dCas12f1-based fusion protein may: form a CRISPR gene-editing system together with a guide RNA; and exhibit various functions relating to a target gene such as base editing and expression control.

This invention provides synthetic exosomes that contain and deliver an effective amount of a gene editor. In certain embodiments the synthetic exosome comprises a liposome formed from a lipid bilayer, where said lipid bilayer comprises: one or more phospholipids selected from the group consisting of phosphate lipids, phosphoglycerol lipids, phosphocholine lipids, and phosphoethanolamine lipids where the lipid carbon chain ranges from 3 to 24 carbon atoms: cholesterol, a cholesterol derivative, or a phytosterol; and a non-ionic surfactant; where the lipid bilayer does not contain an alcohol: the exosome is less than about 200 nm in diameter; and the exosome contains a guide RNA (gRNA) and a cytidine base editor, where the gRNA comprise a sequence that directs said n-Cas9-cytidine deaminase to edit the codon for amino acid 112 of the gene encoding ApoE4.

The present invention provides a method of modifying a targeted site of a double stranded DNA in a host cell, the method including introducing (a) a DNA encoding a crRNA containing a sequence complementary to a target strand of a target nucleotide sequence in the given double stranded DNA, and (b) a DNA encoding a protein group constituting Cascade and a nucleic acid base converting enzyme, in which the nucleic acid base converting enzyme is constituted in a form capable of forming a complex with any protein in the protein group, into the host cell to convert one or more nucleotides in the targeted site to other one or more nucleotides, or delete one or more nucleotides, or insert one or more nucleotides into said targeted site, without cleaving the double stranded DNA in the targeted site.