The present disclosure relates to a novel delivery system with unique modular CRISPR-Cas9 architecture that allows better delivery, specificity and selectivity of gene editing. It represents significant improvement over previously described split-Cas9 systems. The modular architecture is regulatable. Additional aspects relate to systems that can be both spatially and temporally controlled, resulting in the potential for inducible editing. Further aspects relate to a modified viral capsid allowing conjugation to homing agents.

The present disclosure relates to the field of biological technologies, and discloses a method for expressing and preparing a polyvalent multi-specific antibody and an immune hybrid protein. By using the characteristics of protein Intein, a novel method for preparing a polyvalent specific antibody, a hybrid immune protein having an antibody fused to a cytokine, an immunotoxin having an antibody fused to a toxin, or an immune hybrid protein having an antibody fused to other active proteins is designed and developed. Each portion of a hybrid protein is respectively expressed in a suitable prokaryotic or eukaryotic cell system, separated and purified by high-performance affinity chromatography, and then spliced in vitro by trans-splicing mediated by the intein, to prepare a polyvalent specific antibody and an immune hybrid protein. The method has high production efficiency, and wide scope of application, and facilitates the separation and purification of the products.

Methods for producing a trans-splicing intein-modified protease with enhanced solubility and regulating its activity are described. Intein-modified proteases having enhanced solubility and polynucleotides encoding the same are provided. Methods of storing trans-splicing proteases are also described.

The invention relates to proximity-based sortase-mediated protein purification and ligation. Specifically, the invention relates to techniques that links protein expression/purification with conjugation to therapeutic agents, imaging agents, or linkers.

The present disclosure provides a split intein selectable marker system for the production and selection of transgenic cells.

An adenovirus comprising a sequence of formula (I) 5ITR-B.sub.1-B.sub.A-B.sub.2-B.sub.X-B.sub.B-B.sub.Y-B.sub.3-3ITR wherein By comprises a transgene cassette containing four transgenes, said genes encoding a FAP-BITE, CXL10, CXL9, and IFN. The disclosure also extends to a pharmaceutical composition comprising the virus, and use of the virus or formulation in treatment.

Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity of RNA-programmable endonucleases, such as Cas9, or for controlling the activity of proteins comprising a Cas9 variant fused to a functional effector domain, such as a nuclease, nickase, recombinase, deaminase, transcriptional activator, transcriptional repressor, or epigenetic modifying domain. For example, the inventive proteins provided comprise a ligand-dependent intein, the presence of which inhibits one or more activities of the protein (e.g., gRNA binding, enzymatic activity, target DNA binding). The binding of a ligand to the intein results in self-excision of the intein, restoring the activity of the protein.

A cassette sequence for the transformation of a host cell includes at least: a first nucleotide sequence encoding a peptide or protein of interest to be produced by the host cell. The first sequence is linked to a second nucleotide sequence providing resistance to a toxin or encoding an antitoxin peptide to the toxin. The nucleotide sequences are organized in such a way that production of the peptide encoded by the second nucleotide sequence(s) is translationally coupled to production of the peptide encoded by the first nucleotide sequence.

The present disclosure describes a Mtu I-CM intein variant containing one or more mutations or a biologically active fragment thereof, and a method for producing and purifying a molecule of interest using the intein variant. Further described are isolated fusion proteins comprising the intein variant and a tag and a molecule of interest. Also described are expression systems for expressing the intein variant as well as polypeptide screening methods employing the intein variant.

Modified capsid proteins, isolated polynucleotides, methods for the preparation of modified capsid proteins, recombinant viral particles, recombinant expression systems for the generation of modified viral particles, and methods of gene editing and regulation are provided herein.