Provided herein is a circular proximity ligation assay in which proximity-probes are employed as bridges to connect two free oligonucleotides via a dual ligation event, resulting in the formation of a circle. The circles are then quantified by, e.g., qPCR. The addition of an extra oligonucleotide is believed to enhance specificity by decreasing the probability of random background ligation events. In addition, circle formation may have selective advantages, as uncircularized DNA can be removed by a simple exonuclease treatment and it has streamlined the workflow by eliminating preamplification prior to qPCR.

The present invention provides compositions, systems and methods for using ribozyme-mediated cis-cleavage and trans-splicing of RNA molecules to express proteins or fusion proteins of interest.

Provided herein is a circular proximity ligation assay in which proximity-probes are employed as bridges to connect two free oligonucleotides via a dual ligation event, resulting in the formation of a circle. The circles are then quantified by, e.g., qPCR. The addition of an extra oligonucleotide is believed to enhance specificity by decreasing the probability of random background ligation events. In addition, circle formation may have selective advantages, as uncircularized DNA can be removed by a simple exonuclease treatment and it has streamlined the workflow by eliminating preamplification prior to qPCR.

The present disclosure provides a Gin recombinase catalytic domain variant and a zinc finger recombinase comprising a Gin recombinase catalytic domain variant operatively linked to a zinc finger nucleotide binding domain and methods for modifying the genome of a cell or to treat a disorder in a subject by using said zinc finger recombinase protein.

Provided herein is a circular proximity ligation assay in which proximity-probes are employed as bridges to connect two free oligonucleotides via a dual ligation event, resulting in the formation of a circle. The circles are then quantified by, e.g., qPCR. The addition of an extra oligonucleotide is believed to enhance specificity by decreasing the probability of random background ligation events. In addition, circle formation may have selective advantages, as uncircularized DNA can be removed by a simple exonuclease treatment and it has streamlined the workflow by eliminating preamplification prior to qPCR.

The present disclosure provides a polynucleotide comprising an RNA guide sequence, a Cas-binding region, and a DNA template sequence. The disclosure also provides compositions comprising a Cas nuclease or a Cas nickase and one or more polynucleotides comprising a guide sequence, a Cas binding region, and a DNA template sequence. The disclosure further provides a fusion protein comprising a Cas nuclease or a Cas nickase and a DNA polymerase recruitment moiety. Also provided are methods for providing a targeted insertion in a target DNA of a cell.

The present invention pertains to a method for generating a target-specific large serine recombinase (LSR), the method comprising the steps of: a) generating a plurality of first variants of an LSR by introducing one or more mutations into a sequence encoding the LSR; b) generating a library of expression vectors, wherein each expression vector comprises a first region encoding one of the first variants of an LSR, and a second region comprising at least two first target sites of the LSR, wherein the two first target sites are different from each other; c) introducing the library of expression vectors into host cells; d) culturing the host cells and expressing the first variant LSR; e) isolating plasmid DNA from the culture of host cells; f) determining whether a portion of the second region of the expression vector between the two first target sites has been excised by the variant LSR; g) generating a plurality of second LSR variants by introducing one or more mutations into the sequence encoding those first LSR variants that excised the portion of the second region of the expression vector between the two first target sites; and h) repeating steps b) to f) optionally with at least one second target site, wherein the at least one second target site differs from the first target sites in at least one nucleotide. The present invention further provides variant LSRs obtained by the method, a nucleic acid or group of nucleic acids encoding a variant LSR, a vector comprising said nucleic acid(s); a system for integrating a donor DNA into a target nucleic acid; and uses of the LSRs, the nucleic acid(s) or the vector.

This invention provides compositions, reagents, methods, and kits for producing derivatized RNA molecules, particular mRNA molecules encoding a polypeptide and in particular a therapeutic protein, derivatized by linkage to a peptide, aptamer, synthetic DNA or RNA oligonucleotide or molecular probe, capable of targeting the derivatized RNA molecules to a particular subcellular location in a target cell.

Provided is a nucleic acid ligase, which comprises an amino acid sequence having a mutation at one or more positions selected from positions 79, 281, 370 and 372 compared to the amino acid sequence of Hyperligase of the prior art (SEQ ID NO: 1). Also provided are a nucleic acid molecule encoding the enzyme, a vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or the vector. Also provided are a composition containing the enzyme and a use of the enzyme.

Provided herein are compositions, kits, systems, and methods employing single-stranded end-preserving adaptors. Such single-stranded adaptors are attached to DNA duplex molecules while preserving original 5 or 3 single-strand protruding ends (e.g., present in cell-free DNA) by attaching such adapters to 3 ends the DNA duplex molecules using a single strand ligase that has step 3 ligase activity, but not step 2 adenylyl transfer activity, and attaching such adapters to 5 ends of the DNA duplex molecules using a ligase enzyme (e.g., a circligase), thereby forming loop-like structures on one or each end of the DNA duplex molecules. In further embodiments, the loop-like structures are cleaved (e.g., by an endonuclease) as the single-stranded adapters have a cleavable portion, thereby generating a two-part adapter on one or both ends of the DNA duplex molecules that preserves the initial 5 or 3 single-strand protruding ends, along with any methylation present.