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Assay for Oligonucleotides in Serum Without Extraction or RT-PCR

Prof. Ameae Walker’s laboratory at the University of California, Riverside (UCR) has developed an assay to quantify oligos in sub-picomole amounts without the need for sample purification and amplification. This new competitive assay is called an ELOHA (Enzyme-Linked Oligonucleotide Hybridization Assay). The method is illustrated in Fig. 1, below.  Capture Oligos that are to hybridize with an oligo to be measured are covalently linked to a plate (1), a Detection Oligo, with the same sequence as the oligo to be measured, has a conjugated label, such as horseradish peroxidase or biotin.  The Detection Oligo then competes with the oligo of interest for binding to the Capture Oligo (2).  Once the hybridization is complete, the unbound oligos are washed away (3).  A colorimetric readout is produced (4) to inversely quantify the oligo of interest.  Fig. 1 Schematic of the ELOHA assay Fig. 2 shows the use of an ELOHA for amounts of Antimaia in mouse serum. Antimaia is a splice modulating oligomer therapy for breast cancer developed in the UCR lab of Prof. Walker.  

Inhibition Of Stress Granule Formation Through Manipulation Of UBAP2L

Stress granule (SG) formation has been suggested as a two-step process, with initial formation of a dense stable SG ‘‘core’’ followed by accumulation of proteins containing intrinsically disordered regions (IDRs) and low-complexity domains (LCDs) into a peripheral ‘‘shell’’ through a process involving liquid-liquid phase separation (LLPS). Recently, SGs have been associated with human neurodegenerative disorders characterized by the presence of toxic insoluble protein aggregates. This link is most compelling in the case of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), where numerous disease-causing mutations are purported to interfere with LLPS-dependent growth and dynamics of SGs.

Scalable Lipid Bilayer Microfluidics for High-Throughput Gene Editing

Researchers led by Paul Weiss from the Department of Chemistry and Pediatrics at UCLA have created a new microfluidic device for high-throughput gene editing of cells.

Genetically Encoded Aptamer Initiated Fluorescence Complementation (AiFC) Probes For Endogenous RNA Imaging In Live Cells

Direct cellular imaging of the localization and dynamics of biomolecules helps to understand their function and reveals novel mechanisms at the single‐cell resolution. In contrast to routine fluorescent‐protein‐based protein imaging, technology for RNA imaging remains less well explored because of the lack of enabling technology.

Chemical reagents for natural and modified nucleoside triphosphates synthesis

Traditional synthesis of nucleoside triphosphates (NTPs), the building blocks of our genetic material, requires expensive purification yet produces small scale quantities. UCI researchers have developed novel reagents as well as a synthetic route that enables cost-effective and larger scale production of NTPs critical for biomedical research, as well as in certain diagnostic and therapeutic modalities.

“PROTAC-SPARK”: A Genetically-encoded Fluorescent Reporter Platform for Studying PROTAC Interaction Kinetics in Living Cells

This novel class of genetically-encoded fluorescent reporters can be used as powerful tools to study kinetics of protein targeting chimeras (PROTACs) in living cells. These bright, reversible reporters have a large dynamic range and fast kinetics, demonstrating significant advantages over traditional FRET-based fluorescent reporters. This highly-customizable reporter system is ideal for designing novel cell assays to quickly test protein-protein interactions with quantitative results.

Device And Method Of Identifying Exosomes Label-Free

UCLA researchers in the Department of Materials Science and Engineering have developed a new method of using surface enhanced Raman spectroscopy (SERS) with 2D mapping of SERS plus multivariate analysis/machine learning to identify exosomes based on composition and size.

System and Methods for Efficient Collection of Single Cells and Colonies of Cells and Fast Generation of Stable Transfectants

A plate manufactured to enable samples of cells, microorganisms, proteins, DNA, biomolecules, transfectants, and other biological media to be positioned at specific sites. Some or all of the sites are built from removable material so that samples may be isolated.

Efficient Library Preparation for CRISPR Pooled Single-Guide RNAs Screens

There is great interest in both academic and commercial labs in performing pooled CRISPR screens for a variety of purposes, including identifying drug resistance and delivery mechanisms, genes essential for survival, death and disease phenotypes, differentiation, regulation of gene expression, and various other mechanisms.

Single Molecule DNA Profiling

UCLA researchers from the Department of Chemistry and Biochemistry have developed a cutting-edge technique that enables “fingerprinting” of a large number of molecules from a single cell. This technique could revolutionize current molecular diagnostics and prognostics, and therefore lead to personalized medicine in the future.

Endoribonucleases For Rna Detection And Analysis

96 Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin;} Bacteria and archaea possess adaptive immune systems that rely on small RNAs for defense against invasive genetic elements. CRISPR (clustered regularly interspaced short palindromic repeats) genomic loci are transcribed as long precursor RNAs, which must be enzymatically cleaved to generate mature CRISPR-derived RNAs (crRNAs) that serve as guides for foreign nucleic acid targeting and degradation. This processing occurs within the repetitive sequence and is catalyzed by a dedicated CRISPR-associated (Cas) family member in many CRISPR systems.  Endoribonucleases that process CRISPR transcripts are bacterial or archaeal enzymes capable of catalyzing sequence- and structure- specific cleavage of a single- stranded RNA. These enzymes cleave a specific phosphodiester bond within a specific RNA sequence.  UC Berkeley researchers discovered variant Cas endoribonucleases, nucleic acids encoding the variant Cas endoribonucleases, and host cells genetically modified with the nucleic acids that can be used, potentially in conjunction with Cas9, to detect a specific sequence in a target polyribonucleotide and of regulating production of a target RNA in a eukaryotic cell.  For example, it was found that the variant Cas endoribonuclease has an amino acid substitution at a histidine residue such that is is enzymatically inactive in the absence of imidazole and is activatable in the presence of imidazole.  

Generation of Novel Genomic Tools for Use in the Normalization of Endogeneous RNA Expression Between Different Samples

Genome searching tools are a growing field within the medical and biological research communities. There are now a large number of companies offering services relating to understanding genetic information, and typical laboratory functional genomic assays produce a range of data, including sequencing of transcription factors and regulatory regions. Researchers routinely search over 1,417 functional genomic datasets that are publically available, and users have a range of tools to search the data, including many online. Genetic information requires further processing to become biologically meaningful and a pressing challenge is to effectively search functional genomic data and new tools and processes are needed for searching genomic information.

“SPARK (Separation of Phases-based Activity Reporter of Kinase)”_A Genetically-encoded Fluorescent Reporter Platform for Studying Cell Signaling in Living Cells

This novel class of genetically-encoded fluorescent reporters can be used as powerful tools to study protein-protein interactions (PPIs) in living cells. These bright, reversible reporters have a large dynamic range and fast kinetics, demonstrating significant advantages over traditional FRET-based fluorescent reporters.

Small RNA Extraction Kit with High Yield

Prof. Wenwan Zhong and her lab at UCR have developed a method to recover small RNAs using TiO2 fibers which results in a 200-fold improvement in yield when compared to commercially available SiO2 columns. Fig. 1 Schematic of the steps involved in recovering small RNAs using TiO2 fibers and a unique protocol to wash and elute the small RNA.   Fig. 2 Extracting miRNA from MDA-MB-231 cells with TiO2 fibers and SiO2 PureLink miRNA isolation columns. Higher recoveries of endogenous hsa-miR-21 were found with TiO2 fibers when compared with PureLink columns.

High-Throughput Microfluidic Gene-Editing via Cell Deformability within Microchannels

UCLA researchers in the Departments of Pediatrics and Chemistry & Biochemistry have developed a microfluidic device for delivery of biomolecules into living cells using mechanical deformation, without the fouling issues in current systems.

Type V CRISPR/CAS Effector Proteins for Cleaving ssDNA and Detecting Target DNA

Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;} Class 2 CRISPR–Cas systems (e.g., type V CRISPR/Cas systems such as Cas12 family systems) are characterized by effector modules that include a single effector protein. For example, in a type V CRISPR/Cas system, the effector protein - a CRISPR/Cas endonuclease (e.g., a Cas12a protein) - interacts with (binds to) a corresponding guide RNA (e.g., a Cas12a guide RNA) to form a ribonucleoprotein (RNP) complex that is targeted to a particular site in a target nucleic acid via base pairing between the guide RNA and a target sequence within the target nucleic acid molecule.  Thus, like CRISPR-Cas9, Cas12 has been harnessed for genome editing based on its ability to generate targeted, double-stranded DNA (dsDNA) breaks.   UC Berkeley researchers have discovered that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules. The researchers found that target-activated, non-specific ssDNase cleavage is also a property of other type V CRISPR-Cas12 enzymes. By combining Cas12a ssDNase activation with isothermal amplification, the researchers were able to achieve attomolar sensitivity for DNA detection.  For example, rapid and specific detection of human papillomavirus in patient samples was achieved using these methods and compositions.   

Lipoplex-Mediated Efficient Single-Cell Transfection Via Droplet Microfluidics

The invention is an on-chip, droplet based single-cell transfection platform providing higher efficiency and consistency compared to conventional methods. Novel techniques following cell encapsulation yields uniform lipoplex formation, which increases the transfection accuracy. The invention solves the dilemma of the trade-off between efficiency and cell viability, and offers a safe, cell friendly and high transfection solution that is crucial for applications like gene therapy, cancer treatment and regenerative medicine.

A Dual-RNA Guided CasZ Gene Editing Technology

Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;} The CRISPR-Cas system is now understood to confer bacteria and archaea with acquired immunity against phage and viruses. CRISPR-Cas systems consist of Cas proteins, which are involved in acquisition, targeting and cleavage of foreign DNA or RNA, and a CRISPR array, which includes direct repeats flanking short spacer sequences that guide Cas proteins to their targets.  Class 2 CRISPR-Cas systems are streamlined versions in which a single Cas protein bound to RNA is responsible for binding to and cleavage of a targeted sequence. The programmable nature of these minimal systems has facilitated their use as a versatile technology that is revolutionizing the field of genome manipulation, so there is a need in the art for additional Class 2 CRISPR/Cas systems (e.g., Cas protein plus guide RNA combinations).   UC Berkeley researchers discovered a new type of Cas protein, CasZ.  (CasZ) is short compared to previously identified CRISPR-Cas endonucleases, and thus use of this protein as an alternative provides the advantage that the nucleotide sequence encoding the protein is relatively short.  The researchers have shown that the CRISPR CasZ protein and its variants can be used in a complex for specific binding and cleavage of DNA. The CRISPR CasZ complex utilizes a novel RNA and a guide RNA to perform double stranded cleavage of DNA and the complex is expected to have a wide variety of applications in genome editing and nucleic acid manipulation. 

Cas12c/C2C3 Compositions and Methods of Use

96 Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin;} The CRISPR-Cas system is now understood to confer bacteria and archaea with acquired immunity against phage and viruses. CRISPR-Cas systems consist of Cas proteins, which are involved in acquisition, targeting and cleavage of foreign DNA or RNA, and a CRISPR array, which includes direct repeats flanking short spacer sequences that guide Cas proteins to their targets.  Class 2 CRISPR-Cas systems are streamlined versions in which a single Cas protein bound to RNA is responsible for binding to and cleavage of a targeted sequence. The programmable nature of these minimal systems has facilitated their use as a versatile technology that is revolutionizing the field of genome manipulation, so there is a need in the art for additional Class 2 CRISPR/Cas systems (e.g., Cas protein plus guide RNA combinations).   Researchers have shown that a Cas12c protein (also referred to as a Cas12c polypeptide or a C2c3 polypeptide) complex as well as Cas12c variants can be used for specific binding and cleavage of DNA. The Cas12c complex utilizes a novel RNA and a guide RNA to perform double stranded cleavage of DNA. Similar to CRISPR Cas9, Cas12c enzymes are expected to have a wide variety of applications in genome editing and nucleic acid manipulation.   

EpiSort: A Novel Method Using Deep Bisulfite Sequencing to Determine Immune Cell Types in Solid Tissue Samples

EpiSort is a novel method of using DNA methylation patterns to determine the proportion of immune cell populations in solid tissue samples.

An Optimized Active Decapping Complex for Transcription Start Site Mapping

This invention describes an optimized, constitutive active decapping complex from S. pombe for efficient transcription start site (TSS) mapping.

SHARPR-MPRA (Systematic High-Resolution Activation And Repression Profiling With Reporter-Tiling Massively Parallel Reporter Assay)

UCLA researchers in the Department of Biological Chemistry have developed a method to screen hundreds to thousands of genes to identify their regulatory functions.

Integrated Electrowetting Nanoinjector and Aspirator

Gene therapy applications necessitate cell transfection techniques for delivering biomaterial into multiple or a single cell(s). The global market for transfection technologies can be worth more than half a billion by 2017. Current viral and chemical transfection techniques have limited ease of fabrication, transfection efficiency, dosage control, and cell viability. The invention discloses a simple yet efficient technique for nanoinjection of material into a single cell with high transfection efficiency, controlled dosage delivery, and full cell viability.

THERMOSTABLE RNA-GUIDED ENDONUCLEASES AND METHODS OF USE THEREOF (GeoCas9)

96 Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin;} The CRISPR-Cas system is now understood to confer bacteria and archaea with acquired immunity against phage and viruses. CRISPR-Cas systems consist of Cas proteins, which are involved in acquisition, targeting and cleavage of foreign DNA or RNA, and a CRISPR array, which includes direct repeats flanking short spacer sequences that guide Cas proteins to their targets. The programmable nature of these systems has facilitated their use as a versatile technology that is revolutionizing the field of genome manipulation. There is a need in the art for additional CRISPR-Cas systems with improved cleavage and manipulation under a variety of conditions and ones that are particularly thermostable under those conditions.     UC researchers discovered a new type of RNA-guided endonuclease (GeoCas9) and variants of GeoCas9.  GeoCas9 was found to be stable and enzymatically active in a temperature range of from 15°C to 75°C and has extended lifetime in human plasma.  With evidence that GeoCas9 maintains cleavage activity at mesophilic temperatures, the ability of GeoCas9 to edit mammalian genomes was then assessed.  The researchers found that when comparing the editing efficiency for both GeoCas9 and SpyCas9, similar editing efficiencies by both proteins were observed, demonstrating that GeoCas9 is an effective alternative to SpyCas9 for genome editing in mammalian cells.  Similar to CRISPR-Cas9, GeoCas9 enzymes are expected to have a wide variety of applications in genome editing and nucleic acid manipulation.   

Non-Human Primate Adenovirus Model of Human Respiratory Disease

Researchers at the University of California, Davis have developed a model of human respiratory disease using a titi monkey adenovirus.

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