CD39 (CAG>TAG) is one of the most common .sup.0-thalassemic mutation in the Mediterranean area and Latin America, representing >40% of -thalassemic mutations in Tunisia, Argentina and Italy.sup.3. This is a nonsense mutation within the codon of amino acid 39, thus it causes premature translation termination and absence of -globin.sup.4. Here, the inventors exploited adenine base-editors (ABEs) to correct the CD39 (CAG>TAG) mutation in HSPCs from -thalassemia patients and demonstrated the potential of this strategy to correct the pathological phenotype observed during erythroid differentiation. In particular the inventors demonstrated that reverting the CD39 (CAG>TAG) mutation using base editing corrected in vitro the -thalassemic cell phenotype in terms of erythroid differentiation, enucleation, RBC size and apoptosis. The present invention thus relates to base editing approaches for the treatment of -thalassemia, including sickle -thalassemia.

A base editing system for achieving A-to-C and/or A-to-T base mutations and a use thereof are provided. A base editor is constructed by means of fusing 3-methyladenine DNA glycosylase with adenosine deaminase and Cas9 nuclease with impaired catalytic activity, which achieves adenine-based transversion for the first time. It is found through experimental comparison that AXBE, which is constructed by means of fusing mouse-derived 3-methyladenine DNA glycosylase with adenosine deaminase TadA-8e derived from E. coli and Cas9 nickase with impaired catalytic activity derived from Streptococcus pyogenes, has the best effect of catalyzing the transversion of adenine. The use of the base editing system in the gene therapy, cell therapy, human disease model production, and crop genetic breeding, etc. is promoted.

The present disclosure is directed to genetically modified cells that express one or more transgenes at a sustained expression level from a site for safe genomic integration and stable expression. Also provided are methods of making the cells and nucleic acid vectors that can be used to make the cells.

Provided herein are AAV capsid polypeptides comprising peptide modifications relative to the wild-type AAV6 polypeptide that, when present in the capsid of an AAV vector, can facilitate homology directed repair (HDR)-mediated gene editing of human T cells. Also provided are AAV vectors comprising the capsid polypeptides, nucleic acid vectors comprising the encoding nucleic acid molecules, and host cells comprising the vectors, as well as methods of use of such AAV vectors, nucleic acid vectors and host cells.

Compositions, methods, and systems are provided for genome modification of a target sequence in the genome of a cell, using novel engineered Cas endonucleases. These can include a guide polynucleotide/endonuclease system to modify or alter target sequences in the genome of a cell or organism. Also provided are novel effectors and endonuclease systems and elements comprising such systems. Compositions, methods, and systems are also provided that include a guide polynucleotide/endonuclease system comprising at least one endonuclease, optionally covalently or non-covalently linked to, or assembled with, at least one additional protein subunit or substrate.

Provided are gain-of-activity mutations in components of a type I-F3 Tn7-CRISPR-Cas system. The mutations allow use of systems that contain proteins with the mutations to function with both typical and atypical guide RNAs, and permit altered protospacer adjacent motif (PAM) usage.

The present invention is concerned with novel CRISPR-Cas systems which are configured to detect the presence of a target nucleic acid in a sample through activation of secondary nucleases which bind and cleave a nucleic acid probe modified with a (e.g.) fluorophore/quencher moieties, where a change in the property of the probe (e.g. modified fluorescence) reflects the presence of the target nucleic acid in a sample to be tested.

Described herein are compositions, systems, and methods for modulating gene expression. Also described herein are systems and methods for treating a disease or a condition by modulating gene expression. In some embodiments, the compositions, systems, and methods provided herein are used to treat familial hypercholesterolemia. In some embodiments, the compositions, systems, and methods provided herein involve suppression of endogenous PCSK9.

The present invention provides method of killing first cells (for example pathogenic cells) which are slow-growing under stress conditions (for example, stationary phase cells, stressed cells, persister cells, biofilm cells and dormant cells) by providing a vector encoding a toxic agent or component thereof under the control of a stress-phase active (SPA) promoter. The invention also provides methods of expressing proteins of interest (POIs) in such slow-growing cells under stress conditions, which POIs may be therapeutic molecules and reporter genes for example, by providing a vector encoding a POI under the control of a SPA promoter. There are provided methods of treatment using such vectors.

The present disclosure provides improved systems and methods for production of bacteriophage-based engineered transducing particles and uses thereof in manipulating bacterial populations to express any nucleic acid sequence of interest. The disclosed systems are based on inserting to producing cells nucleic acid molecules that comprise the nucleic acid sequence of interest and a protective regulated array, and regulators specific for the regulator array. The system further provides helper transducing particle that facilitates the propagation of the transducing particle.