The invention relates to peptide linkers and fusion proteins comprising linkers designed for optimizing the activity of the proteins comprised therein, and methods for using the same. The invention further relates to newly designed Cas12a-based adenine base editors.

Provided herein are compositions of engineered guide RNAs relating to providing or engineering a structural target to attract adenosine deaminase acting on RNA (ADAR) editing to a desired site and methods of use thereof. Further provided herein are compositions and methods relating to recombinant adenosine deaminase acting on RNA (ADAR) split guide RNAs (adar-sgRNA). In certain embodiments, the adar-sgRNA composition comprises two guides-one guide with an ADAR recruiting domain and a second guide with a 5′ and/or 3′ RNA targeting domain for forming a trimolecular complex at a mismatch site that serves to recruit ADAR. Binding of the two adar-split gRNAs to the target RNA forms a trimolecular complex which recruits ADAR enzymes to deaminate one or more mismatched adenosine residues in the adar-split gRNA RNA targeting domain: target RNA duplex. In certain embodiments, such compositions and methods will be useful for modifying a coding sequence of a desired protein.

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.

The invention provides compositions comprising novel adenosine base editors (e.g., ABE8) that have increased efficiency and methods of using these adenosine deaminase variants for editing a target sequence and methods of using same to treat genetical disorder or conditions, e.g. sickle cell disease, with engraftment.

Disclosed are a LEAPER technique-based nucleic acid drug and a method for treating diseases such as Hurler syndrome by means of using the nucleic acid drug to target and edit RNA. The method comprises: performing an editing from adenosine base to hypoxanthine base on RNA by using the nucleic acid drug to precisely repair the pathogenic G>A mutation site of, for example, Hurler syndrome; thereby recovering the normal in vivo expression of a protein encoded by RNA, such as IDUA.

Provided herein are methods of delivering “split” Cas9 protein or nucleobase editors into a cell, e.g., via a recombinant adeno-associated virus (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 dnaEn and dnaEc, respectively). Upon co-expression, the two sections of the Cas9 protein or nucleobase editor are ligated together via intein-mediated protein splicing. Recombinant AAV vectors and particles for the delivery of the split Cas9 protein or nucleobase editor, and methods of using such AAV vectors and particles are also provided.

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 invention relates to peptide linkers and fusion proteins comprising linkers designed for optimizing the activity of the proteins comprised therein, and methods for using the same. The invention further relates to newly designed Cas12a-based adenine base editors.

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.