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 invention relates to a method of site-directed integration into a genetic locus encoding at least one activity of the pyrimidine salvage pathway in a host cell, wherein said activity of the pyrimidine salvage pathway is purine/cytosine permease (FcyB), cytosine deaminase (FcyA), uracil-phosphoribosyl-transferase (Uprt), concentrative nucleoside transporter (CntA) or uridine kinase (UK), comprising: a) providing a host cell comprising a functional copy of the genetic locus encoding at least one activity of the pyrimidine salvage pathway; (b) introducing a gene or sequence of interest into said host cell via transformation of an integrative nucleic acid construct which comprises 3′ and/or 5′ of the gene or sequence of interest flanks being homologous to said genetic locus or which carries a sequence being homologous to said genetic locus of the pyrimidine salvage pathway and thus allowing for a homologous recombination at said genetic locus, wherein said homologous recombination is capable of causing an inactivation or reduction of the activity encoded by said genetic locus; (c) growing a transformed host cell under selective medium conditions, wherein said medium comprises an efficient amount of 5-flucytosine (5-FC), 5-fluorouracil (5-FU) or 5-fluorouridine (5-FUR); and (d) selecting a host cell which is capable of growing under the medium conditions of step (c). Also envisaged is a host cell, comprising at least one gene or sequence of interest in one or more genetic loci encoding an activity of the pyrimidine salvage pathway wherein said gene or sequence of interest replaces or partially replaces the sequence encoding said at least one activity of the pyrimidine salvage pathway at said locus, the use of such a host cell for the production of several activities, as well as the use of a genetic locus encoding at least one activity of the pyrimidine salvage pathway in a host cell in a process of transforming said host cell or a process of genetically modifying said host cell.

Provided is a method for modulating RNA splicing by inducing a base mutation at a splice site or a base substitution in a polypyrimidine region. The method comprises expressing a targeting cytosine deaminase in a cell, to induce AG at a 3′ splice site of an intron of interest in a gene of interest to mutate into AA, or to induce GT at a 5′ splice site of the intron of interest in a gene of interest to mutate to AT, or to induce a plurality of Cs in a polypyrimidine region of the intron of interest in a gene of interest to respectively mutate into Ts. The method specifically blocks an exon recognition process, modulates a selective splicing process of endogenous mRNA, induces exon skipping, activates an alternative splice site, induces mutually exclusive exon conversion, induces intron retention, and enhances an exon.

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 cytosine base editors.

A method is disclosed for decreasing or retarding an increase in the size of a localized or metastatic tumor by using a combination of an immune stimulating cytotoxic gene therapy and immune-checkpoint modulating agent, in conjunction with other therapies, including radiation therapy, chemotherapy, surgery, and immunotherapies.

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.

The invention provides pluripotent cells that are used therapeutically for regenerating tissues but avoid rejection by subjects that receive them. In particular, the invention provides hypo-immunogenic pluripotent cells that avoid host immune rejection. The cells lack major immune antigens that trigger immune responses and are engineered to avoid phagocytic endocytosis. The invention further provides universally acceptable “off-the-shelf” pluripotent cells and derivatives thereof for generating or regenerating specific tissues and organs.

Provided herein are systems, compositions, and methods of introducing protective and/or loss-of-function variants of CCR5 and CCR2. Variants may be introduced using a CRISPR/Cas9-based nucleobase editor or other guide nucleotide sequence-programmable DNA binding protein domain-based fusion protein described herein. Further provided herein are compositions and methods of preventing and treating conditions related to HIV infection and progression as well as to AIDS.

The present disclosure generally relates to a synthetic therapeutic bacteriophage displaying at least one therapeutic agent, wherein the at least one therapeutic agent is fused to a coating protein of the synthetic bacteriophage.

Disclosed herein are CRISPR/Cas-based base editing compositions and methods for treating Duchenne Muscular Dystrophy by restoring dystrophin function.