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 comprise a Gam protein, a napDNAbp, and a cytidine deaminase. In some embodiments, the fusion proteins further comprise a UGI domain. 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 Gam protein, a cytidine deaminase and nucleic acid editing proteins or domains, are provided.

The invention features compositions and methods for modifying a polynucleotide (e.g., DNA) using a nucleobase editor comprising a first DNA binding protein domain that is catalytically inactive, a domain having base editing activity, and a second CDNA binding protein domain having nickase activity. The invention also features a fusion protein comprising a domain having base editing activity (e.g., cytidine deaminase or adenosine deaminase), and two nucleic acid programmable DNA binding protein domains (napDNAp), a first napDNAbp comprising nickase activity and a second napDNAbp that is catalytically inactive, where at least the two napDNAbps are joined by a linker, as well as related methods for using such base editors, and kits comprising the base editors.

The invention provides a method of modifying a targeted site of a double stranded DNA, including a step of contacting a complex wherein a nucleic acid sequence-recognizing module that specifically binds to a target nucleotide sequence in a selected double stranded DNA and a nucleic acid base converting enzyme are linked, with the 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 the targeted site, without cleaving at least one strand of the double stranded DNA in the targeted site.

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.

MAGE-A1 specific T cell receptors (TCRs) are provided. Accordingly, there is provided a TCR comprising a TCR α chain as set forth in SEQ ID NO: 1 having at least one mutation at an amino acid position selected from the group consisting of S189, G125, W55 and Y56; and/or a TCR β chain as set forth in SEQ ID NO: 2 having at least one mutation at an amino acid position selected from the group consisting of S32, S109 and T63, the TCR binds a MAGE-A1 peptide as set forth in SEQ ID NO: 25. Also provided are polynucleotides encoding the TCR and T cells expressing same and methods of use 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.

The present disclosure provides nucleobase editors that include a cytidine deaminase domain, a codon-optimized nuclease-defective Cas9 domain, and at least one nuclear-localization sequence. The nucleobase editors disclosed herein improve the efficiency by which single-nucleotide variants can be created compared to conventional BE3 nucleobase editors.

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.

The invention, in some aspects, includes systems, methods and components of molecular recorders that encode the timing of transcriptional activity into the sequence of RNA, which can then enable a sequencing-based readout of the internal dynamics of cells.

Provided herein are methods and compositions for highly precise base editing and single strand nicking. In particular, provided herein are methods for producing a genetically modified cell where the methods employ a universal, highly precise base editor or staggered Cas9 editor for precise base editing with minimal off-target or bystander effects.