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 invention provides a miniaturized cytidine deaminase-containing complex for modifying DNA formed by combining a nucleic acid sequence recognition module and cytidine deaminase, wherein the nucleic acid sequence recognition module specifically binds to a target nucleotide sequence of double-stranded DNA, the cytidine deaminase is composed of an amino acid sequence composed of a region of amino acid residues at positions 30-150 of SEQ ID NO: 1, an ortholog thereof, an amino acid sequence having mutations of one or several amino acids therein, or an amino acid sequence having at least 90% similarity thereto, and the targeted site of the double-stranded DNA is modified.

The present invention belongs to the field of gene editing. In particular, the present invention relates to an improved cytosine base editing system which has a significantly reduced genome-wide off target effect and a narrow editing window.

A duchenne muscular dystrophy-related exonic splicing enhancer, sgRNA and gene editing tool can be applied in the preparation of drugs for treating duchenne muscular dystrophy. The gene editing tool designed on the basis of cytosine deaminase AID mutants and Cas9 mutants can perform site-specific modification on a mammalian genome by using an adeno-associated virus (AAV) as a vector. By optimizing an encoding nucleic acid sequence and an element composition structure of the editing tool, site-specific targeted modification of mammalian genetic material DNA can be efficiently achieved; and by performing targeted genetic manipulation on the nucleic acid sequence carrying disease mutations, a pathogenic mutation cannot be retained in a mature protein amino acid sequence or the pathogenic mutation cannot perform its function, so that the purpose of treating various gene mutation type genetic rare diseases is achieved, and the advantages of high efficiency, safety and stability are achieved.

The instant specification provides for evolved base editors which overcome deficiencies of those in art (including increased efficiency and/or decreased requirement for specific sequence-context at an editing site) and which are obtained a result of a phage-assisted continuous evolution (PACE) system. In particular, the instant specification provides for evolved cytidine base editors (e.g., based on APOBEC1, CDA, or AID cytidine deaminase domains) which overcome deficiencies of those in art (including increased efficiency and/or decreased requirement for specific sequence-context at an editing site) and which are obtained a result of a phage-assisted continuous evolution (PACE) system.

The clinical history of β-hemoglobinopathies shows that the severity is mitigated by the synthesis of the fetal γ-globin in adulthood, typically associated with genetic variants the HBB cluster known as hereditary persistence of fetal hemoglobin (HPFH) mutations. The inventors identified that most of the known HPFH mutations in the γ-globin promoters (C>T, G>A, T>C or A>G) can be recapitulated using CBE- and ABE-mediatedbase-editing approaches. In particular, the inventors designed gRNAs that, when combined with CBEs or ABEs, generate HPFH mutations, and either disrupt binding sites for transcriptional repressors (-200 and -115 sites) or generate de novo DNA motifs recognized by transcriptional activators (e.g., -198 T>C, the -175 T>C and -113 A>G). It is noteworthy that a subset of the gRNAs targeting the -200 and the 115 regions are predicted to generate simultaneously HPFH mutations and also to make base changes other than HPFH mutations in or around the LRF and BCL11A binding sites, which might further reduce LRF and BCL11A occupancy. Accordingly, the present invention relates to base editing approaches for the treatment of β-hemoglobinopathies.

The present invention provides novel systems, methods and compositions for making and using a recombinantly engineered novel Cas9 optimized for human cells, for nucleic acid targeting and manipulation. The present invention is based on the discovery of a novel Cas9 species from Lachnospira bacterium that was codon-optimized and recombinantly produced for use in human ceils. In some embodiments, the novel Cas9 can be used in a base editor. In some embodiments, the novel engineered Cas9 is used to treat human diseases.

Described herein are systems, methods, and compositions capable of targeting nucleic acids. Describe in certain exemplary embodiments herein are a class of small Cas proteins (Type II-D Cas proteins) and systems thereof. Also described in certain exemplary embodiments herein are methods of modifying target sequences using the class of small Cas proteins (Type II-D Cas proteins) and systems thereof described herein.

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 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.