The invention relates to fusion proteins which comprise at least one antigenic amino acid sequence fused to a carrier heterologous protein sequence, wherein the antigenic sequence comprises an epitopic sequence of a Plasmodium protein and the carrier heterologous protein sequence is a sequence that is immunogenic in humans. The proteins are useful as anti-malaria vaccines.

The present disclosure is directed to a lysin-AMP polypeptide construct comprising: (a) a first component comprising the polypeptide sequence of: (i) SEQ ID NO: 118 (GN202); or (ii) a polypeptide having lysin activity and having at least 80% sequence identity with the polypeptide sequence of SEQ ID NO: 118 (GN202); or (iii) an active fragment of SEQ ID NO: 118 (GN202); and (b) a second component comprising the polypeptide sequence of at least one antimicrobial peptide (AMP), wherein the at least one AMP comprises SEQ ID NO: 114 (FIRL). Exemplary lysin-AMP polypeptides, such as GN370 (SEQ ID NO: 44) as well as methods of treating bacterial infections using the present lysin-AMP polypeptide constructs are also disclosed.

The present disclosure is directed to a lysin-AMP polypeptide construct comprising: (a) a first component comprising the polypeptide sequence of: (i) SEQ ID NO: 118 (GN202); or (ii) a polypeptide having lysin activity and having at least 80% sequence identity with the polypeptide sequence of SEQ ID NO: 118 (GN202); or (iii) an active fragment of SEQ ID NO: 118 (GN202); and (b) a second component comprising the polypeptide sequence of at least one antimicrobial peptide (AMP), wherein the at least one AMP comprises SEQ ID NO: 114 (FIRL). Exemplary lysin-AMP polypeptides, such as GN370 (SEQ ID NO: 44) as well as methods of treating bacterial infections using the present lysin-AMP polypeptide constructs are also disclosed.

The invention concerns a trans-splicing RNA (tsRNA) molecule comprising one or multiple unstructured binding domains; a cell or vector comprising said tsRNA; and a method for killing cells or treating a disease using said tsRNA.

The invention concerns a trans-splicing RNA (tsRNA) molecule comprising one or multiple unstructured binding domains; a cell or vector comprising said tsRNA; and a method for killing cells or treating a disease using said tsRNA.

The invention relates to a nucleic acid comprising at least one methylation-protectable restriction element, the methylation-protectable restriction element comprising: (i) a type IIS restriction enzyme recognition sequence, or a partial type IIS restriction enzyme recognition sequence, that is recognised by a type IIS restriction enzyme that cleaves outside of the recognition sequence; (ii) a DNA methylase recognition sequence that is recognised and methylated by a DNA methylase, wherein the DNA methylase recognition sequence is identical to, or is encompassed within, the type IIS restriction recognition sequence, such that methylation of the nucleic acid by the DNA methylase methylates the type IIS restriction enzyme recognition sequence and protects the nucleic acid from cleavage by the type IIS restriction enzyme; and (iii) a recognition sequence for a sequence-specific DNA-binding protein, wherein the recognition sequence is positioned such that the binding of the sequence-specific DNA-binding protein overlaps with the DNA methylase recognition sequence such that binding of the sequence-specific DNA-binding protein is capable of preventing methylation of the type IIS restriction enzyme recognition sequence by the DNA methylase such that it is not protected from cleavage by the type IIS restriction enzyme. The invention further relates to associated methods of nucleic acid assembly.

The invention relates to a nucleic acid comprising at least one methylation-protectable restriction element, the methylation-protectable restriction element comprising: (i) a type IIS restriction enzyme recognition sequence, or a partial type IIS restriction enzyme recognition sequence, that is recognised by a type IIS restriction enzyme that cleaves outside of the recognition sequence; (ii) a DNA methylase recognition sequence that is recognised and methylated by a DNA methylase, wherein the DNA methylase recognition sequence is identical to, or is encompassed within, the type IIS restriction recognition sequence, such that methylation of the nucleic acid by the DNA methylase methylates the type IIS restriction enzyme recognition sequence and protects the nucleic acid from cleavage by the type IIS restriction enzyme; and (iii) a recognition sequence for a sequence-specific DNA-binding protein, wherein the recognition sequence is positioned such that the binding of the sequence-specific DNA-binding protein overlaps with the DNA methylase recognition sequence such that binding of the sequence-specific DNA-binding protein is capable of preventing methylation of the type IIS restriction enzyme recognition sequence by the DNA methylase such that it is not protected from cleavage by the type IIS restriction enzyme. The invention further relates to associated methods of nucleic acid assembly.

The type V-U1 system from the bacterium Mycobacterium mucogenicum CCH10-A2 (Mmu) has a nuclease which binds dsDNA but it does not cleave it. Additionally, after dsDNA binding by the nuclease an RuvC-dependent interference of nascent transcript (mRNA) takes place and this mechanism has not been described before for any CRISPR system. CRISPR based gene manipulation can therefore use CRISPR endonucleases from the Type V-U1 system from Mycobacterium mucogenicum, including variant and modified endonucleases, so as to provide for methods of expression control and gene editing in cells of any living organism or of any nucleic acid in vitro.

The type V-U1 system from the bacterium Mycobacterium mucogenicum CCH10-A2 (Mmu) has a nuclease which binds dsDNA but it does not cleave it. Additionally, after dsDNA binding by the nuclease an RuvC-dependent interference of nascent transcript (mRNA) takes place and this mechanism has not been described before for any CRISPR system. CRISPR based gene manipulation can therefore use CRISPR endonucleases from the Type V-U1 system from Mycobacterium mucogenicum, including variant and modified endonucleases, so as to provide for methods of expression control and gene editing in cells of any living organism or of any nucleic acid in vitro.

Provided is a method for knocking in a gene of interest to a cell. The genome of the cell contains a negative selectable marker, e.g., a thymidine kinase gene flanked by a pair of recombinase recognition sites (RRS), e.g., attP. The method involves introducing into the cell a targeting construct that contains a gene of interest flanked by a second pair of RRS, e.g., attB. The targeting construct also contains in the vector backbone a negative selectable marker, e.g., thymidine kinase gene. When a recombinase recognizing the RRS is expressed, the recombination events between the two pairs of RRS result in the site-specific integration of the gene of interest in the genome of the cell. Upon selection based on the negative selectable marker, the parental cells, cells with undesired integration, e.g., random integration, or the integration of the vector backbone are removed.