Abstract: The present disclosure provides compositions and methods for making and using methanotrophic proline auxotrophs.

Abstract: Therapeutic methods for cancer treatments using a combined prokaryotic-eukaryotic delivery and expression system for the delivery of multiple therapeutic factors via a modified tumor-targeted bacteria. A targeted bacteria-vector system elicits an inter-kingdom dual expression (IKDE) of antitumor agents, in the nucleus or cytoplasm of eukaryotic cells, with priming and maintenance of the vector in the bacterium. The therapeutic factors include small interfering RNAs, tumoricidal proteins, DNA molecules, or a combination thereof. The system provides direct killing of tumor cells and alters the tumor microenvironment by expressing anti-angiogenic factors and cytokines in intracellular and/or extracellular environments. Also provided are methods of using natural exosomes comprising cargoes obtained from the bacterially infected cells. The bacteria-vector system is useful for many types of tumor and cancer as well as recombinant vaccines.

Abstract: The present disclosure provides a DNA-targeting RNA that comprises a targeting sequence and, together with a modifying polypeptide, provides for site-specific modification of a target DNA and/or a polypeptide associated with the target DNA. The present disclosure further provides site-specific modifying polypeptides. The present disclosure further provides methods of site-specific modification of a target DNA and/or a polypeptide associated with the target DNA The present disclosure provides methods of modulating transcription of a target nucleic acid in a target cell, generally involving contacting the target nucleic acid with an enzymatically inactive Cas9 polypeptide and a DNA-targeting RNA. Kits and compositions for carrying out the methods are also provided. The present disclosure provides genetically modified cells that produce Cas9; and Cas9 transgenic non-human multicellular organisms.

Abstract: The present disclosure provides a DNA-targeting RNA that comprises a targeting sequence and, together with a modifying polypeptide, provides for site-specific modification of a target DNA and/or a polypeptide associated with the target DNA. The present disclosure further provides site-specific modifying polypeptides. The present disclosure further provides methods of site-specific modification of a target DNA and/or a polypeptide associated with the target DNA The present disclosure provides methods of modulating transcription of a target nucleic acid in a target cell, generally involving contacting the target nucleic acid with an enzymatically inactive Cas9 polypeptide and a DNA-targeting RNA. Kits and compositions for carrying out the methods are also provided. The present disclosure provides genetically modified cells that produce Cas9; and Cas9 transgenic non-human multicellular organisms.

Abstract: The present disclosure provides a DNA-targeting RNA that comprises a targeting sequence and, together with a modifying polypeptide, provides for site-specific modification of a target DNA and/or a polypeptide associated with the target DNA. The present disclosure further provides site-specific modifying polypeptides. The present disclosure further provides methods of site-specific modification of a target DNA and/or a polypeptide associated with the target DNA The present disclosure provides methods of modulating transcription of a target nucleic acid in a target cell, generally involving contacting the target nucleic acid with an enzymatically inactive Cas9 polypeptide and a DNA-targeting RNA. Kits and compositions for carrying out the methods are also provided. The present disclosure provides genetically modified cells that produce Cas9; and Cas9 transgenic non-human multicellular organisms.

Abstract: The present disclosure provides a DNA-targeting RNA that comprises a targeting sequence and, together, with a modifying polypeptide, provides for site-specific modification of a target DNA and/or a polypeptide associated with the target DNA. The present disclosure further provides site-specific modifying polypeptides. The present disclosure further provides methods of site-specific modification of a target DNA and/or a polypeptide associated with the target DNA The present disclosure provides methods of modulating transcription of a target nucleic acid in a target cell, generally involving contacting the target nucleic acid with an enzymatically inactive Cas9 polypeptide and a DNA-targeting RNA. Kits and compositions for carrying out the methods are also provided. The present disclosure provides genetically modified cells that produce Cas9; and Cas9 transgenic non-human multicellular organisms.

Abstract: Provided are oligonucleotides that are capable of detecting KRAS and PIK3CA mutations in both cancer patients and healthy individuals with high specificity in kPCR assays. When the oligonucleotides are used as forward primers in conjunction with a defined genotyping algorithm spreadsheet, the primers are capable of enhancing detection of KRAS codon 12, 13, and 61 and PIK3CA codon 542, 545, and 1047 single nucleotide polymorphisms (SNPs) in a background of wild-type sequences. The oligonucleotides of the present invention are also capable of preventing pseudogene amplification when the oligonucleotides are hybridized as reverse primers or detection probes to the mismatch sequences.

Abstract: A clustered regularly interspaced short palindromic repeat (CRISPR)-associated complex for adaptive antiviral defence (Cascade); the Cascade protein complex comprising at least CRISPR-associated protein subunits Cas7, Cas5 and Cas6 which includes at least one subunit with an additional amino acid sequence possessing nucleic acid or chromatin modifying, visualising, transcription activating or transcription repressing activity. The Cascade complex with additional activity is combined with an RNA molecule to produce a ribonucleoprotein complex. The RNA molecule is selected to have substantial complementarity to a target sequence. Targeted ribonucleoproteins can be used as genetic engineering tools for precise cutting of nucleic acids in homologous recombination, non-homologous end joining, gene modification, gene integration, mutation repair or for their visualisation, transcriptional activation or repression.

Abstract: Methods and compositions for bacterial production of pure single-stranded DNA (ssDNA) composed of custom sequence and size have been developed. The methods enable scalability and bio-orthogonality in applications of scaffolded DNA origami, offering one-step purification of large quantities of pure ssDNA amendable for immediate folding of DNA nanoparticles. The methods produce pure ssDNA directly from bacteria. In some embodiments the E. coli helper strain M13cp combined with a phagemid carrying only an f1-origin allows for, without the need for additional purification from contaminating dsDNA. This system is useful for generalized circular ssDNA synthesis, and here is applied to the assembly of DNA nanoparticles folded both in vitro and direct from phage.

Abstract: This invention relates to processes that transcribe DNA molecules containing non-standard nucleotides using variants of T7 RNA polymerase to give RNA transcripts that contain their complementary non-standard nucleotides. Non-standard nucleotides pair during transcription using patterns of hydrogen bonding that are different from patterns that join the thymine-adenine and guanine-cytosine nucleobase pairs.

Abstract: The present invention provides a method of modifying a targeted site of a double stranded DNA, including a step of contacting a complex wherein a nucleic acid sequence-recognizing module that specifically binds to a target nucleotide sequence in a selected double stranded DNA and DNA glycosylase with sufficiently low reactivity with a DNA having an unrelaxed double helix structure (unrelaxed DNA) are bonded, with the double stranded DNA, to convert one or more nucleotides in the targeted site to other one or more nucleotides or delete one or more nucleotides, or insert one or more nucleotides into the targeted site, without cleaving at least one strand of the double stranded DNA in the targeted site.

Abstract: The present disclosure provides a DNA-targeting RNA that comprises a targeting sequence and, together with a modifying polypeptide, provides for site-specific modification of a target DNA and/or a polypeptide associated with the target DNA. The present disclosure further provides site-specific modifying polypeptides. The present disclosure further provides methods of site-specific modification of a target DNA and/or a polypeptide associated with the target DNA The present disclosure provides methods of modulating transcription of a target nucleic acid in a target cell, generally involving contacting the target nucleic acid with an enzymatically inactive Cas9 polypeptide and a DNA-targeting RNA. Kits and compositions for carrying out the methods are also provided. The present disclosure provides genetically modified cells that produce Cas9; and Cas9 transgenic non-human multicellular organisms.

Abstract: There is provided a non-PCR based method for construction of a DNA recombination fragment with necessary flanking regions homologous to a desired site of genetic manipulation within a target DNA required for recombination-based manipulation of said target DNA, comprising the steps of: A) identifying a desired site of insertion for a genetic element in said target DNA; B) locating an endogenous, native, half-site of a selected restriction endonuclease present upstream of the site within the DNA to be targeted for genetic manipulation and thereby defining the 5? extent of an upstream homology region; C) locating an endogenous, native, half-site of the same selected restriction endonuclease present downstream of the site within the DNA to be targeted for genetic manipulation and thereby defining the 3? extent of a downstream homology region; D) synthesising a flanking region cassette resulting in juxtapositioning of the upstream and downstream half-sites thereby causing formation of complete restriction site for

Abstract: Provided herein are constructs for genome editing or genetic engineering its fungi or protists, methods of using the constructs and media for use in selecting cells. The construct include a polynucleotide encoding a thymidine kinase operably connected to a promoter, suitably a constitutive promoter; a polynucleotide encoding an endonuclease operably connected to an inducible promoter; and a recognition site for the endonuclease. The constructs may also include selectable markers for use in selecting recombinations.

Abstract: An in vitro method for screening for candidate compounds for preventing and/or attenuating skin ageing, and/or hydrating skin, includes: a) contacting a test compound with a sample of papillary fibroblasts; b) measuring the expression of a gene selected from PDPN, CCRL1 and NTN1, in the papillary fibroblasts; and c) selecting compounds for which an activation of at least 1.5 fold of the expression of at least one of the genes is measured in the treated papillary fibroblasts compared with untreated papillary fibroblasts. Another in vitro method includes: a) contacting a test compound with a sample of reticular fibroblasts; b) measuring the expression of a gene selected from MGP, PPP1R14A and TGM2, in the reticular fibroblasts; and c) selecting compounds for which an activation of at most 1.0 fold of the expression of at least one of the genes is measured in the treated reticular fibroblasts compared with untreated reticular fibroblasts.

Abstract: A method for producing recombinant adeno-associated virus, the method including: (1) transforming a gene of interest (GOI) into a recombinant baculovirus, where the recombinant baculovirus has a genome integrated with AAV Rep gene, Cap gene, and rAAV genome ITR-GOI that are needed in the production of the rAAV; and where the ITR-GOI is linked to expression cassette of the Cap gene and the Rep gene by a 5? terminal nucleic acid segment or a 3? terminal nucleic acid segment; (2) infecting host insect larvae with the recombinant baculovirus prepared in (1), such that the rAAV is produced in vivo in the host insect larvae; and (3) lysing the host insect larvae obtained in (2), and extracting and purifying the rAAV.

Abstract: Methods, compositions, and kits are provided herein for CRISPER/Cas-mediated nucleic acid detection or modification.

Abstract: The present disclosure provides a DNA-targeting RNA that comprises a targeting sequence and, together with a modifying polypeptide, provides for site-specific modification of a target DNA and/or a polypeptide associated with the target DNA. The present disclosure further provides site-specific modifying polypeptides. The present disclosure further provides methods of site-specific modification of a target DNA and/or a polypeptide associated with the target DNA The present disclosure provides methods of modulating transcription of a target nucleic acid in a target cell, generally involving contacting the target nucleic acid with an enzymatically inactive Cas9 polypeptide and a DNA-targeting RNA. Kits and compositions for carrying out the methods are also provided. The present disclosure provides genetically modified cells that produce Cas9; and Cas9 transgenic non-human multicellular organisms.

Abstract: The present disclosure provides a DNA-targeting RNA that comprises a targeting sequence and, together with a modifying polypeptide, provides for site-specific modification of a target DNA and/or a polypeptide associated with the target DNA. The present disclosure further provides site-specific modifying polypeptides. The present disclosure further provides methods of site-specific modification of a target DNA and/or a polypeptide associated with the target DNA The present disclosure provides methods of modulating transcription of a target nucleic acid in a target cell, generally involving contacting the target nucleic acid with an enzymatically inactive Cas9 polypeptide and a DNA-targeting RNA. Kits and compositions for carrying out the methods are also provided. The present disclosure provides genetically modified cells that produce Cas9; and Cas9 transgenic non-human multicellular organisms.

Abstract: The present invention relates to a method to detect the interaction between a target ligand and a moiety of interest using an adenylate cyclase enzyme (AC) and calmodulin (CaM) as interacting partners, said method comprising: i) expressing in a suitable host cell: (a) a low number of molecules of a first chimeric polypeptide containing AC, and (b) a low number of molecules of a second chimeric polypeptide containing CaM, wherein said AC in said first chimeric polypeptide and/or said CaM in said second chimeric polypeptide has decreased affinity for its interacting partner, wherein said AC in said first chimeric polypeptide is fused to a moiety of interest and said CaM in said second chimeric polypeptide is fused to a target ligand, or conversely, and wherein, when said moiety of interest and said target ligand interact, said AC is activated, and ii) detecting the activation of said AC.