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 invention provides a method for engineering B lymphocytes by utilizing activation-induced cytidine deaminase of the B lymphocyte. Thereby, use of engineered nucleases, such as Cas nuclease, can be avoided. Engineered B cells are useful to produce customized antibodies and for B cell therapy. Accordingly, the present invention also provides engineered B cells and customized antibodies produced by engineered B cells.

Provided are fusion proteins and related molecules useful for conducting base editing with reduced or no off-target mutations. The fusion protein may include a first fragment comprising a nucleobase deaminase or a catalytic domain thereof, a second fragment comprising a nucleobase deaminase inhibitor, 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.

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.

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.

Some aspects of this disclosure relate to strategies, systems, methods, compositions, and kits that are useful for production (e.g., evolution) of cytidine deaminase protein variants that are characterized by increased soluble expression and/or stability relative to the wild-type cytidine deaminase protein from which they are evolved. In some embodiments, evolved cytidine deaminase variants described by the disclosure are useful for incorporation into targeted nucleic acid editing proteins, for example in fusion proteins with a Cas9 domain or variant thereof.

The present invention belongs to the field of plant genetic engineering. Specifically, the invention relates to a method for creating novel herbicide resistant plants by base editing techniques and a method for screening endogenous gene mutation sites capable of conferring herbicide resistance in plants. The invention also relates to the use of the identified endogenous gene mutantation sites in crop breeding.

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. The disclosure provides fusion proteins of nucleic acid programmable DNA binding proteins (napDNAbp), e.g., Cas9 or variants thereof, and nucleic acid editing proteins such as cytidine deaminase domains (e.g., novel cytidine deaminases generated by ancestral sequence reconstruction), and adenosine deaminases that deaminate adenine in DNA. Aspects of the disclosure relate to fusion proteins (e.g., base editors) that have improved expression and/or localize efficiently to the nucleus. In some embodiments, base editors are codon optimized for expression in mammalian cells. In some embodiments, base editors include multiple nuclear localization sequences (e.g., bipartite NLSs), e.g., at least two NLSs. In some embodiments, methods for targeted nucleic acid editing are provided.

The present invention provides a method for modifying a targeted site of a double-stranded DNA in a cell, the method including a step of bringing a complex in which a nucleic acid sequence-recognizing module that specifically binds to a selected target nucleotide sequence in a double-stranded DNA and a nucleic acid base converting enzyme or DNA glycosylase are linked, and a donor DNA containing an insertion sequence into contact with said double-stranded DNA, to substitute the targeted site with the insertion sequence, or insert the insertion sequence into said targeted site, without cleaving at least one strand of said double-stranded DNA in the targeted site.

Compositions and methods for genome editing of Bacteroides species are provided herein. RNA-guided nucleobase modification systems are engineered to target specific loci in chromosomal DNA of a target bacteria cell, wherein the genome of the target bacterial cell can be modified.