The invention features compositions and methods for conditioning a patient (e.g., to facilitate transplantation and/or engraftment). The invention provides a base editing strategy targeting cell surface proteins that is useful for conditioning. In one aspect, the invention provides methods of producing a hematopoietic stem cell or progenitor thereof for the treatment of a hemoglobinopathy, hematologic cancer, or myeloproliferative disease.

The invention features compositions and methods for conditioning a patient (e.g., to facilitate transplantation and/or engraftment). The invention provides a base editing strategy targeting cell surface proteins that is useful for conditioning. In one aspect, the invention provides methods of producing a hematopoietic stem cell or progenitor thereof for the treatment of a hemoglobinopathy, hematologic cancer, or myeloproliferative disease.

Provided herein are systems, compositions, and methods for the incorporation of unnatural amino acids into proteins via nonsense suppression or rare codon suppression. Nonsense codons and rare codons may be introduced into the coding sequence of a protein of interest using a CRISPR/Cas9-based nucleobase editor described herein. The nucleobase editors are able to be programmed by guide nucleotide sequences to edit the target codons in the coding sequence of the protein of interest. Also provided are application enabled by the technology described herein.

Provided herein are systems, compositions, and methods for the incorporation of unnatural amino acids into proteins via nonsense suppression or rare codon suppression. Nonsense codons and rare codons may be introduced into the coding sequence of a protein of interest using a CRISPR/Cas9-based nucleobase editor described herein. The nucleobase editors are able to be programmed by guide nucleotide sequences to edit the target codons in the coding sequence of the protein of interest. Also provided are application enabled by the technology described herein.

Some aspects of this disclosure provide compositions, 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 capable of inducing a cytosine (C) to guanine (G) change in a nucleic acid (e.g., genomic DNA) are provided. In some embodiments, fusion proteins of a nucleic acid programmable DNA binding protein (e.g., Cas9) and nucleic acid editing proteins or protein domains, e.g., deaminase domains, polymerase domains, and/or base excision enzymes 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 a nucleic acid programmable DNA binding protein (e.g., Cas9), and nucleic acid editing proteins or domains, are provided.

Some aspects of this disclosure provide compositions, 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 capable of inducing a cytosine (C) to guanine (G) change in a nucleic acid (e.g., genomic DNA) are provided. In some embodiments, fusion proteins of a nucleic acid programmable DNA binding protein (e.g., Cas9) and nucleic acid editing proteins or protein domains, e.g., deaminase domains, polymerase domains, and/or base excision enzymes 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 a nucleic acid programmable DNA binding protein (e.g., Cas9), and nucleic acid editing proteins or domains, are provided.

The present invention provides an isolated nucleic acid molecule comprising, or consisting of, the nucleic acid sequence of SEQ ID NO:1 or of a nucleic acid sequence of at least 1400 bp having at least 80% identity to said sequence of SEQ ID NO:1, wherein said isolated nucleic acid molecule leads to the specific expression of an exogenous gene in agmat-positive cells in layer ⅔ of mouse cortex when a nucleic acid sequence coding for said exogenous gene is operatively linked to said isolated nucleic acid molecule.

The present invention provides a method for producing a fermentation product by culturing recombinant exosporium-producing Bacillus cells that express a fusion protein of interest on the exosporium in a medium containing sources of carbon and nitrogen in a total concentration of at least 20 g/L, resulting in a fermentation broth containing a high titer of the recombinant Bacillus spores.

The present invention provides a method for producing a fermentation product by culturing recombinant exosporium-producing Bacillus cells that express a fusion protein of interest on the exosporium in a medium containing sources of carbon and nitrogen in a total concentration of at least 20 g/L, resulting in a fermentation broth containing a high titer of the recombinant Bacillus spores.

Engineered transversion base editors that enable expanded amino acid modifications and methods of using the same. Described herein, for example, are fusion proteins containing cytidine deaminases (e.g. human or rat APOBECs, pmCDA1 or AID) or adenosine deaminases (e.g. E. coli TadAs) or a combination thereof, catalytically impaired CRISPR-Cas proteins (e.g. Cas9, CasX or Cas12 nucleases), linkers, nuclear localization signals (NLSs) and a human or E. coli uracil-n-glycosylase (UNG) and/or REV1 protein that enable the CRISPR-guided programmable introduction of C-to-G and G-to-C transversions in DNA. The UNG may be fused to the deaminase-Cas fusion or not, in which case endogenous UNG may be recruited using molecular machinery that is integrated into the deaminase-Cas fusion architecture, e.g. using peptide or RNA aptamers or scFVs, sdABs or Fabs.