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Composition and Methods of a Nuclease Chain Reaction for Nucleic Acid Detection

This invention leverages the nuclease activity of CRISPR proteins for the direct, sensitive detection of specific nucleic acid sequences. This all-in-one detection modality includes an internal Nuclease Chain Reaction (NCR), which possesses an amplifying, feed-forward loop to generate an exponential signal upon detection of a target nucleic acid.Cas13 or Cas12 enzymes can be programmed with a guide RNA that recognizes a desired target sequence, activating a non-specific RNase or DNase activity. This can be used to release a detectable label. On its own, this approach is inherently limited in sensitivity and current methods require an amplification of genetic material before CRISPR-base detection. 

COMPOSITIONS AND METHODS FOR IDENTIFYING HOST CELL TARGET PROTEINS FOR TREATING RNA VIRUS INFECTIONS

Viral infection is a multistep process involving complex interplay between viral life cycle and host immunity. One defense mechanism that hosts use to protect cells against the virus are nucleic-acid-mediated surveillance systems, such as RNA interference-driven gene silencing and CRISPR-Cas mediated gene editing. Another important stage for host cells to combat virus replication is translational regulation, which is particular important for the life cycle of RNA viruses, such as Hepatitis C virus and Coronavirus.  While efforts to characterize structural features of viral RNA have led to a better understanding of translational regulation, no systematical approaches to identify important host genes for controlling viral translation have been developed and little is known about how to regulate host-virus translational interaction to prevent and treat infections caused by RNA viruses.   UC Berkeley researchers have developed a high-throughput platform using CRISPR-based target interrogation to identify new therapeutics targets or repurposed drug targets for blocking viral RNA translation.  The new kits can also be used to identify important domains within target proteins that are required for regulating (viral RNA translation) and can inform drug design and development for treating RNA viruses.

Anti-Acinetobacter Baumannii Polyclonal Antibody (AB-pAb)

The inventors have constructed a polyclonal antibody (pAb) for the specific detection of the multi-drug resistant (MDR) bacterial pathogen, Acinetobacter baumannii (A. baumannii), producing the antibody entitled, 'AB-pAb'. The AB-pAb was raised against a recombinant (His-tagged) 22 kDa outer membrane protein (OMP22), an antigenic protein which is conserved across the species. The gene encoding OMP22 was amplified from the clinical A. baumannii isolate, AR_0056, which belongs to the international clonal lineage II, a lineage associated with outbreaks worldwide. The AB-pAb is capable of recognizing purified, denatured, OMP22 by Western blot, in addition to the native protein in whole cells of A. baumannii in vitro. The pAb was optimized for diagnostic use by, firstly, removing antibodies within the heterogeneous pAb pool which were cross-reactive to other, clinically-relevant Gram-negative bacteria (GNB). This eliminates the issue of cross-reactivity often associated with polyclonal antibodies, which can limit their use as diagnostic tools. Moreover, testing was performed under conditions which mimic those of the blood and urine, further enhancing the novel AB-pAb's ability to recognize target bacteria in patient samples. When tested against a panel of clinical isolates by indirect-ELISA, for the recognition of A. baumannii from other clinically relevant GNB, the optimized AB-pAb had a sensitivity of 85.5% (95 % confidence interval: 76.11% to 92.3%) and a specificity of 99.5% (95 % confidence interval: 99.53% to 99.99%) at a cutoff, signal-to-noise ratio (SNR) of 0.1275. To our knowledge, no commercial anti-A. baumannii pAbs are currently available which target OMP22, specifically optimized for diagnostic purposes.

Chimeric Cas9 Variants With Novel Engineered Enzymatic Activities

In this invention, the HNH domain of a Cas9 is replaced by a domain that could have diverse enzymatic activities. This invention enables engineering of Cas9 chimeras that possess novel, conformation-sensitive enzymatic activity to perform specific genome editing in vitro, in vivo, and ex vivo.Prior to this invention, all of the strategies to engineer Cas9 fusion proteins and provide Cas9 with non-natural enzymatic activity for genome manipulations were engineered by fusing specific domains to the N- or C-terminus of Cas9 via long and flexible linkers, or through domain insertion approach. The disadvantages of these synthetic Cas9 chimeras are that the attached domain is on the long flexible linker, and it is very dynamic. Thus, these fusions have a broad activity window and they are large, which makes it difficult to deliver them to the cells. 

Decorating Chromatin for Precise Genome Editing Using CRISPR

A novel fusion construct that fuses Cas9 to a truncated version of human PRDM9 with the purpose of improving precise genome editing via homologous direceted repair (HDR). PRDM9 is a protein that deposits histone marks H3K4me3 and H3K36me3 simultaneously during meiosis to mark recombination hot spots where crossover occurs and is resolved by homologous recombination. H3K36me3 has also been demonstrated to be required upstream of homologous recombination repair after double stranded breaks (DSBs) and during V(D)J recombination for adaptive immunity. Recent evidence suggests PRDM9 acts as a pioneer factor opening closed chromatin. The newly engineered PRDM9C-Cas9 fusion construct shows increased HDR and decreased non-homologous end joining mediated insertions and deletions (indels).

Small Cas9 Protein Inhibitor

A new protein that is able to inhibit the Cas9 protein from Streptococcus iniae (SinCas9). SinCas9 is capable of robust DNA cleavage and offers an immune orthogonal Cas9 for use in gene editing in human cells. The inhibitor is a small protein from a phage and is capable of inhibiting SinCas9 activity in vitro and in human cell genome editing experiments.

Novel Phage CRISPR-Cas Effectors and Uses Thereof

UC Berkeley researchers have discovered a novel family of proteins denoted Cas12L within the Type V CRISPR Cas superfamily distantly related to CasX, CasY and other published type V sequences.  These Cas12L proteins utilize a guide RNA to perform RNA-directed cleavage of DNA.

Single Conjugative Vector for Genome Editing by RNA-guided Transposition

The inventors have constructed conjugative plasmids for intra- and inter-species delivery and expression of RNA-guided CRISPR-Cas transposases for organism- and site-specific genome editing by targeted transposon insertion. This invention enables integration of large, customizable DNA segments (encoded within a transposon) into prokaryotic genomes at specific locations and with low rates of off-target integration.

Multiphoton Magnetic Resonance Imaging

UC Berkeley researchers have developed novel imaging techniques with the use of a multiphoton magnetic resonance imaging apparatus. By taking a particular rotating frame transformation the researchers found that multiphoton excitations appear just like single‐photon excitations and can also use concepts explored in standard single‐photon excitation. One prototype included a low frequency coil while another prototype included no additional hardware but instead used oscillating gradients as a source of extra photons for excitation.  The methods and multiphoton MRI can be used to transform a standard slice selective adiabatic inversion pulse into a multiband version without modifying the RF pulse itself. The addition of oscillating gradients creates multiphoton resonances at multiple spatial locations and allows for adiabatic inversions at each location.

Cellular Potassium Imaging Using A Ratiometric Fluorescent Sensor

The inventors developed a ratiometric fluorescent small molecule probe for potassium ion detection composed of a duo-fluorophore system (KR-1). UV-vis detector and fluorometer measurement support ratiometric response of the probe towards potassium ion concentration. The probe was further applied to cellular potassium level detection using confocal microscope imaging technique. KR-1 enables simple determination of potassium levels in various cancer or non-cancer cell lines.

Covalent Organic Framework With Exceptional Water Sorption Properties

A new covalent organic framework (COF) with defective square lattice topology and exceptional water sorption properties stemming fro its unique framework structure. The COF exhibits a working capacity of 0.23 g(H2O)/g(COF) between 20 and 40% relative humidity without displaying hysteretic behavior. Furthermore, it maintains these promising water sorption properties after several uptake and release cycles. This material could be used as a sorbent for water harvesting or other water sorption related applications.

Improved Cas12a Proteins for Accurate and Efficient Genome Editing

Mutated versions of Cas12a that remove its non-specific ssDNA cleavage activity without affecting site-specific double-stranded DNA cutting activity. These mutant proteins, in which a short amino acid sequence is deleted or changed, provide improved genome editing tools that will avoid potential off-target editing due to random ssDNA nicking.

DEVICES AND METHODS FOR GENERATING OLIGODENDROCYTE PROGENITOR CELLS

The emergence of several cell based therapy candidates in the clinic is an encouraging sign for human diseases/disorders that currently have no effective small molecule or biologic based therapy. Stem cells – including adult and pluripotent subtypes – offer tremendous clinical promise for the treatment of a variety of degenerative diseases, as these cells have the capacity to self-renew indefinitely and to mature into functional cell types and thereby serve as a source of cell replacement therapies (CRTs) and pluripotent stem cells (hPSCs) are of increasing interest for the development of CRTs because of their capacity to differentiate into all cell types in an adult, for which adult tissue-specific stem cells may in some cases not even exist. One potential CRT enabled by hPSCs is oligodendrocyte progenitor cells (OPCs) for the treatment of spinal cord injury (SCI). Such hPSC-OPCs have recently advanced to a Phase II clinical trial and are even being considered for additional diseases in the central nervous system (CNS), such as multiple sclerosis (MS), or injury from radiation.   UC researchers have developed a microscale 3D culture screening and analysis methodology that is relevant to the production of several up and coming cell replacement therapy candidates for which derivation from a precursor cell type requires searching through a large in vitro design space of doses, durations, dynamics, and combinations of signaling cues over several weeks of culture, such as oligodendrocyte progenitor cells (OPCs) and midbrain dopaminergic neurons (mDA neurons) derived from human pluripotent stem cells. 

Strongly Interacting Magnetic Particle Imaging

Nuclear medicine is a diagnostic imaging method that works very well, but it is both expensive and gives off excess radiation. X-rays also are used for diagnostic imaging but have poor contrast. Magnetic Particle Imaging (MPI)is a promising new tracer modality with zero attenuation in tissue, near-ideal contrast and sensitivity, and an excellent safety profile, however, the spatial resolution of MPI is currently the modality’s only weak technical attribute. UC Berkeley and UF researchers have developed a novel, compact, and intuitive MPI scanner that resolves this issue.  The research demonstrated proof-of-concept studies for an MPI modality, referred to herein as strongly-interacting magnetic particle imaging (siMPI) that enables a super-resolution breakthrough. The siMPI provided more than a 6-fold improvement in every dimension of space spatial resolution and 37-fold increase in sensitivity. The MPI can be used for early-stage detection of cancer, gut bleeds, strokes, pulmonary embolism, and tracking immunotherapies and MPI can penetrate any tissue, including bone, lungs, and dense breast tissue.

CRISPR-CAS EFFECTOR POLYPEPTIDES AND METHODS OF USE THEREOF

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 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.  Current CRISPR Cas technologies are based on systems from cultured bacteria, leaving untapped the vast majority of organisms that have not been isolated.  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 12 protein.  Site-specific binding and/or cleavage of a target nucleic acid (e.g., genomic DNA, ds DNA, RNA, etc.) can occur at locations (e.g., target sequence of a target locus) determined by base-pairing complementarity between the Cas12 guide RNA (the guide sequence of the Cas12 guide RNA) and the target nucleic acid.  Similar to CRISPR Cas9, Cas12 enzymes are expected to have a wide variety of applications in genome editing and nucleic acid manipulation.    

Targeted Ionophore-Based Metal Supplementation

Metal deficiency is implicated in a variety of genetic, neurological, cardiovascular, and metabolic diseases. Current approaches for addressing metal deficiency rely on generic metal ion supplementation, which can potentially lead to detrimental off-target metal accumulation in unwanted tissues and subsequently trigger oxidative stress and damage cascades. The inventors have developed a new modular platform for delivering metal ions in a tissue-specific manner and demonstrate liver-targeted copper supplementation as a proof of concept of this strategy. Specifically, the inventors designed and synthesized a N-acetylgalactosamine-functionalized ionophore, Gal-Cu(gtsm), to serve as a copper-carrying “Trojan Horse” that targets liver-localized asialoglycoprotein receptors (ASGPRs) and releases copper only after being taken up by cells, where the reducing intracellular environment triggers copper release from the ionophore. The inventors utilized a combination of bioluminescence imaging and inductively-coupled plasma mass spectrometry assays to establish ASGPR-dependent copper accumulation with this reagent in both liver cell culture and mouse models with minimal toxicity. The modular nature of this synthetic approach presages that this platform can be expanded to deliver a broader range of metals to specific cells, tissues, and organs in a more directed manner to treat metal deficiency in disease. This patent broadly covers directed metal delivery to select organs, tissues, and organelles.

Simultaneous Detection Of Protein Isoforms And Nucleic Acids From Low Starting Cell Numbers

Embryo-specific nucleic acid modifications, including retrotransposon activity-derived genomic modifications and alternative splicing of mRNA, is crucial for the development of mammalian embryos. However, determining if all genomic modifications and mRNA isoforms translate to protein variations remain intriguing questions due to difficulty in measuring protein isoforms and nucleic acids from small starting cell numbers.    UC Researchers have developed a system for performing dual nucleic acid and protein isoform measurements on low starting cell numbers equivalent to the number of blastomeres composing early embryonic development stages (morula and blastocysts).  The system integrates fractionation polyacrylamide gel electrophoresis (fPAGE) with off-chip analysis of nucleic acids in the nuclei. An additional method can be used to remove nuclei for off-chip analysis. The system can measure expression of protein isoforms from the cytoplasmic fraction of 1-100 cells while achieving analysis of either DNA or mRNA retained in the nuclei. The researchers have demonstrated signal from immunoprobed protein correlates strongly with protein expression prior to lysis in TurboGFP-expressing cells and that mRNA levels correlate with protein abundance in TurboGFP-expressing cells.

A Protein Inhibitor Of Cas9

  Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 nucleases, when complexed with a guide RNA, effect genome editing in a sequence-specific manner. RNA-guided Cas9 has proven to be a versatile tool for genome engineering in multiple cell types and organisms.  There is a need in the art for additional compositions and methods for controlling genome editing activity of CRISPR/Cas9.   UC Berkeley researchers have discovered a new protein that is able to inhibit the Cas9 protein from Staphyloccocus aureus (SauCas9). SauCas9 is smaller than the frequently used Cas9 from Streptococcus pyogenes, which has a number of benefits for delivery. The inhibitor is a small protein from a phage and is capable of strongly inhibiting gene editing in human cells.

NANOPORE MEMBRANE DEVICE AND METHODS OF USE THEREOF

Several chemical, physical, and biological techniques have been used for delivering macromolecules into living cells. Delivery of biomolecules into living cells is essential for biomedical research and drug development as well as genome editing. However, conventional methods of delivery of biomolecules such as viral vectors, cell penetrating peptides, cationic lipids, positive charged polymers, bulk electroporation, and microinjection pose several challenges. Such challenges include safety concerns, toxicity, damage to the cells, limited loading capacity, low delivery efficiencies, low cell viabilities, low cell throughput, high cellular perturbation, and high costs.  Thus, there is a need for delivery devices and methods that allow for permeabilization of the cell membrane to facilitate delivery of biomolecules into cells.   UC Berkeley researchers have developed a universal delivery electroporation system that makes cell transfection very simple for all of types of cells. The technology can be used to replace conventional cellular delivery methods such as cationic lipid, positive charged polymer and bulk electroporation as well as microinjection.  The system can deliver biomolecules (e.g., DNA, RNA, proteins, nucleic acid-protein complexes (e.g., RNPs)) or other reagents into all cell types, including T-cells, which cannot be efficiently transfected with conventional approaches.  

Bioinspired Hydrogels for the Treatment of Volumetric Muscle Loss Injury

Injuries that involve a degree of muscle tissue loss that exceeds the endogenous regenerative capacity of muscle, resulting in permanent cosmetic and functional deficits of either the injured muscle or the muscle unit, are referred to as volumetric muscle loss (VML) injuries. Current treatment for VML injury involves surgical muscle transfer, although these procedures are often associated with poor engraftment and donor site morbidity.    UC Berkeley and U.Va researchers have developed a new technology for the treatment of VML injuries that overcomes the limitations associated with current treatments for VML injury.  The Matrix Assisted Cell Transplantation (MACT) technology developed by the researchers employs “bioinspired” materials designed to emulate regulatory processes that modulate cell function in the stem/progenitor cell microenvironment.  The technology includes: 1) peptide ligands to imitate the natural extracellular matrix (ECM); 2) proteolytic remodeling via matrix metalloproteinase (MMP) sensitive peptide crosslinks; and, 3) growth factors with engineered density and presentation.    The technology and the materials used have been shown to significantly improve donor survival after transplantation, promote angiogenesis, and encourage donor cell integration with the host tissue.

Cas12-mediated DNA Detection Reporter Molecules

Class 2 CRISPR-Cas systems are streamlined versions in which a single Cas protein (an effector protein, e.g., a type V Cas effector protein such as Cpf1) 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 continues to revolutionize the field of genome manipulation.    Cas12 is an RNA-guided protein that binds and cuts any matching DNA sequence. Binding of the Cas12-CRISPR RNA (crRNA) complex to a matching single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) molecule activates the protein to non-specifically degrade any ssDNA in trans. Cas12a-dependent target binding can be coupled to a reporter molecule to provide a direct readout for DNA detection within a sample.  UC Berkeley researchers have developed compositions, systems, and kits having labeled single stranded reporter DNA molecules that provide a sensitive readout for detection of a target DNA. 

Tissue Projection Electrophoretic Separation Of Protein

A range of related immunoblotting methods have enabled the identification and semi-quantitative characterization of e.g., DNA (Southern blot), RNA (northern blot), proteins (Western blot), and protein-protein interactions (far-western blot); by coupling biomolecule separations and assays.  However, there are a wide number of alternative splicing events, post-translational modifications, and co-translational modifications (e.g., phosphorylation, glycosylation, and protein cleavage) that give rise to proteoforms and protein complexes with distinct function and subsequent cell behavior that cannot be analyzed with conventional methods such as immunohistochemistry (IHC). Analytical variability (lack of isoform- or complex-specific antibody probes), biological variability (small cell subpopulations diluted in bulk analysis), and lack of multiplexing (measurement of multiple proteins from the same tissues) can all render proteoforms and protein complexes undetectable by current technologies.     UC Berkeley researchers have created electrophoretic separation platform that is capable of measuring proteoforms and protein complexes lacking specific antibodies alongside spatial information, at the cellular level.  This platform maintains the architecture of 2D tissue slices while projecting a protein separation in the 3rd dimension. The platform mitigates artifacts induced by tissue dissociation processes, as the intact tissue is lysed and subject to a protein separation. The platform is also compatible with differential detergent fractionation methods for further separation of proteins (e.g. separation by localization within the cell, by cell type, by protein complex formation, or by cellular vs. matrix proteins), opening the door for a novel, refined classification taxonomy using enhanced biomarker signatures for diagnostics and treatment selection in oncology among a wide range of additional future applications.  

Puromycin Activity-Based Sensing Probes For Molecular Imaging And Histochemistry

A novel class of puromycin activity-based sensing probes containing analyte-specific responsive triggers have been synthesized and utilized for molecular imaging and histochemistry. After specific reaction between the trigger on the probe and target analyte, free puromycin molecules will be released and incorporated into nascent peptides. These incorporated puromycin can be detected after immunostaining, thus offering a highly sensitive method for detection of target analytes due to no leakage problem (as found in some reported fluorescent probes) and high signal-to-noise level from immunostaining. The syntheses of the probes are highly versatile, and representative examples for detection of reactive oxygen species (ROS), reactive sulfur species (RSS), reactive carbonyl species (RCS), ROS scavengers, and redox active metal ions have been demonstrated. One exemplary probe is Peroxymycin-1, which contains H2O2-responsive aryl boronate conjugated to puromycin through carbamate linkage. Peroxymycin-1 shows robust performance on molecular imaging of H2O2 in cell culture and histochemical analysis of H2O2 level in tissue samples harvested from small animals. It has been further employed for detection of elevated H2O2 level in liver tissues from a murine model of non-alcoholic fatty liver disease (NAFLD), suggesting its potential for studying disease pathology associated with H2O2 as well as disease diagnosis and monitoring of treatment progress.

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.  

Printed All-Organic Reflectance Oximeter Array

A flexible reflectance oximeter array (ROA) composed of printed organic light-emitting diodes (OLEDs) and organic photodiodes (OPDs), which senses reflected light from tissue to determine the oxygen saturation. Since reflected light is used as the signal, the sensor array can be used beyond the conventional sensing locations. We implemented the ROA to measure SpO2 on the forehead with 1.1% mean error and to create two-dimensional (2D) oxygenation maps of the adult forearm under pressure cuff-induced ischemia. Due to the mechanical flexibility, 2D oxygenation mapping capability, and the ability to place the sensor in diverse places, the ROA is promising for novel medical sensing applications such as mapping oxygenation in tissues, wounds, or transplanted organs.

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