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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.  

Enhanced Speed Polymerases For Sanger Sequencing

Sanger sequencing has remained a dominant DNA sequencing methodology for molecular biology research and development for many years.  The main commercially available DNA polymerase developed for Sanger sequencing has a slow extension speed and has difficulties sequencing secondary structures such as GC rich regions, hairpins, mono- and poly-nucleotide repeats.  While specialized plastics and reductions in reaction volumes to improve Sanger sequencing reaction times, any gains in sequencing assay performance (e.g., sequencing time or throughput) are offset by increased costs associated with a terminator reagent.  During the last two decades, further refinement and advancement of suitable DNA polymerases to improve polymerization speeds during Sanger sequencing have been limited.  Thus, there remains a need for improved DNA polymerases suitable for Sanger sequencing that possess enhanced elongation speeds, and the ability to sequence through secondary structures present in DNA templates.    A UC Berkeley researchers has discovered compositions and methods for preparing and using Taq DNA polymerases with improved Sanger sequencing elongation sequencing rates as compared to commercially available Sanger sequencing reagents.  

Protein-Coated Microparticles For Protein Standardization In Single-Cell Assays

Single-cell analysis offers powerful capabilities of identification of rare sub-populations of cells, understanding heterogeneity of cancerous tumors, and tracking cell differentiation and reprogramming. Despite great potentials for uncovering new biological systems and targeting diseases with precision medicine, single-cell approaches are composed of complex device processes that can cause bias in measurement.  In deep sequencing, technical variation in single cell expression data occurs during capture and pre-amplification steps. Similarly, in single-cell protein assays, technical variability can obscure functionally relevant variance.    To better control protein measurement quality in single-cell assays, researchers at the University of California, Berkeley developed a novel method to loading and release protein standard. This method utilizes the surface of modified and functionalized microparticles as vehicles to capture target proteins with desired concentrations. Chelation-assisted click chemistry is applied to demonstrate that protein standards with different molecular masses can be loaded and bounded in a single-cell protein assay. Microparticles are introduced into single-cell devices by either passive gravity, magnetic attraction, or other physicochemical forces. These protein standards from microparticles provide a reference to measure protein mass sizes from individual cells and a quality control for any biases in device fabrication, cell lysis, protein solubility, protein capture, and protein readouts (i.e. antibody probing).   

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.  

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.   

Class 2 CRISPR/Cas 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 Class 2 CRISPR Cas protein and their variants can be used in a complex for specific binding and cleavage of DNA. The Class 2 CRISPR Cas 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. 

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. 

CRISPR CASY 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).   Previously UC Berkeley researchers discovered a new type of Cas protein, CasY (also referred to as Cas 12d protein).  CasY 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.  CasY utilizes a guide RNA to perform double stranded cleavage of DNA. The researchers introduced CRISPR-CasY into E. coli, finding that they could block genetic material introduced into the cell.  Further research results indicated that CRISPR-CasY operates in a manner analogous to CRISPR-Cas9, but utilizing an entirely distinct protein architecture containing different catalytic domains.   CasY is also expected to function under different conditions (e.g., temperature) given the environment of the organisms that CasY was expressed in.  Similar to CRISPR Cas9, CasY enzymes are expected to have a wide variety of applications in genome editing and nucleic acid manipulation. Recent studies have shown that the CasY complex utilizes a novel RNA, in addition to the guide RNA, to perform double stranded cleavage of DNA. Similar to CRISPR Cas9, CasY enzymes are 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.   

Modulation Of Wnt5a To Treat Glaucoma

A major risk factor for glaucoma which affects over 3 million Americans and 60 million people worldwide is increased intraocular pressure (IOP), which can damage the optic nerve and cause permanent blindness without treatment. Currently, there is no cure for glaucoma. Existing eye drops or oral medications are of limited efficacy with many side effects, and surgeries often fail with scar formation and fibrosis. Schlemm’s canal (SC) is a circumferential channel located at the iridocorneal angle in the ocular anterior chamber. It is part of the conventional aqueous humor outflow system, which accounts for 70–90% of the total aqueous humor that drains out of the eye in human. The endothelial cell lining of Schlemm’s canal is one of the primary sites of resistance to aqueous humor drainage and is a major determinant of IOP. When canal resistance increases with age or under pathological situation, IOP is elevated leading to glaucoma with irreversible optic nerve damage and vision loss. It is therefore an important target for glaucoma therapy.    UC researchers have discovered that Wnt5a is expressed on Schlemm’s canal, where its expression is regulated in response to sheer stress change, and devised a method for treating Glaucoma or pathogenic intraocular pressure by locally administering to an eye in need thereof formulations of a Wnt5a inhibitor.

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.   

Gene Delivery Into Mature Plants Using Carbon Nanotubes

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;} Current methods of biomolecule delivery to mature plants are limited due to the presence of plant cell wall, and are additionally hampered by low transfection efficiency, high toxicity of the transfection material, and host range limitation. For this reason, transfection is often limited to protoplast cultures where the cell wall is removed, and not to the mature whole plant.  Unfortunately, protoplasts are not able to regenerate into fertile plants, causing these methods to have low practical applicability. Researchers at the University of California have developed a method for delivery of genetic materials into mature plant cells within a fully-developed mature plant leaf, that is species-independent. This method utilizes a nano-sized delivery vehicle for targeted and passive transport of biomolecules into mature plants of any plant species. The delivery method is inexpensive, easy, and robust, and can transfer biomolecules into all phenotypes of any plant species with high efficiency and low toxicity.

Xylosyl-Xylitol Oligomers And Their Microbial And Enzymatic Productions

Lignocellulosic biomass derived from plant cell walls is the most abundant raw material for biofuels and renewable chemicals production.  Hemicellulose comprises about 30% of the total weight of lignocellulosic biomass. In contrast to cellulose, hemicellulose components are readily depolymerized into short oligomers and released into the liquid phase during pretreatment.  It is of great interest to convert the released hemicellulose components into fuels or other value-add chemicals for building an economical biomass conversion process. There are ten times more microorganisms than human cells in a healthy adult.  The symbiosis between the microbiome and human organs is increasingly recognized as a major player in health and well-being.  Xylooligosaccharides and xylitol, both derived from hemicellulose, can benefit gut flora and oral flora, respectively. Xylooligosaccharides (XOS, also called xylodextrins) are naturally occurring oligosaccharides, found in bamboo shoots, fruits, vegetables, milk and honey.  Industrial scale production of XOS can be carried out with much less expensive lignocellulosic materials by hydrothermal treatment or enzymatic hydrolysis.  A broad range of applications of XOS have been demonstrated, including as functional food, prevention and treatment of gastrointestinal infections, animal feed for fish and poultry, agricultural yield enhancer and ripening agent, and as active agents against osteoporosis, pruritus cutaneous, otitis, and skin and hair disorders.  In the current market, the most important applications of XOS correspond to ingredients for functional foods as a prebiotic, or formulated as synbiotics. XOS has been shown to promote beneficial bacteria Bifidobacterium adolescentis growth in vitro and in vivo.  It has been estimated that the prebiotics market will reach $4.8 billion by 2018. Xylitol is another hemicellulose-derived compound beneficial to human health.  For many bacteria and yeasts, the uptake of non-utilizable xylitol interferes with hexose utilization, which helps the human body to rebuild a healthy microbiome.  Xylitol has been used to prevent middle ear infections and tooth decay.  In addition, xylitol possesses 33% fewer calories but similar sweetness compared to sucrose and has been widely used as a substitute sweetener.  While chemical hydrogenation of xylose remains the major industrial method of xylitol production, microbial fermentation has become more popular in the newly built plants due to lower conversion cost. There exists a need for improved methods of producing xylooligosaccharides and related compounds, such as xylooligosaccharides with xylitol components.    UC researchers discovered a new set of fungal metabolic intermediates, named xylosyl-xylitol oligomers and developed the enzymatic and microbial fermentation method to produce such compounds. The detection and purification methods have also been developed.

Mechano-Nps (Node Pore Sensing)

The mechanical properties of cells derive from the structure and dynamics of their intracellular components, including the cytoskeleton, cell membrane, nucleus, and other organelles.  These, in turn, emerge from cell specific genetic, epigenetic, and biochemical programs, providing a link between cellular mechanics and the underlying molecular state.  Differences in mechanical properties reflect on cellular properties with clinical implications, including the metastatic potential, cell-cycle stage, and differentiation state of cells.  Yet, many mechanical aspects of various cells and sub-cell organelles remain unknown due to absence of appropriate analysis platforms. Atomic-force microscopy (AFM) and micropipette aspiration are the gold standards for performing mechanical measurements of cells, as they both provide controlled loading conditions and quantify such cellular properties as elastic modulus and cortical tension.  They are, however, burdened by slow throughput, capable of analyzing only just a few cells/hr.  Likewise, optical tweezers and microplate rheometry also suffer from low throughput.  Various microfluidic based platforms have been proposed for the high-throughput mechanical analysis of cells, including hydrodynamic stretching cytometry, suspended microchannel resonators (SMR), and real-time deformability cytometry (RT-DC).  Although each of these methods can analyze populations of cells in a relatively short time, they focus only on a single cellular property.  Consequently, these platforms, and the low-throughput traditional methods that under-sample, can neither identify cellular heterogeneity nor classify mechanical sub-phenotypes within a population. Investigators at UC Berkeley have developed a microfluidic platform, “mechano-node-pore sensing” (mechano-NPS), a rapid and multi-parametric cell screening platform, that simultaneously quantifies cell diameter, transit time through a contraction channel, transverse deformation under constant strain, and recovery time after deformation.  This platform efficiently reveals malignant-dependent mechanical phenotypes of cancer and normal epithelial cells, discriminates between sub-lineages of cells with accuracy comparable to flow cytometry, and determines the effects of chronological age and malignant progression on cell elasticity and recovery from deformation – based solely on a cell’s mechanical properties.

Method For Imaging Neurotransmitters In Vitro and In Vivo Using Functionalized Carbon Nanotubes

Neurotransmitters play a central role in complex neural networks by serving as chemical units of neuronal communication.  Quantitative optical methods for the detection of changes in neurotransmitter levels has the potential to profoundly increase our understanding of how the brain works. Therapeutic drugs that target neurotransmitter release are used ubiquitously to treat a vast array of brain and behavioral disorders.  For example, new methods in this sphere could provide a new platform by which to validate the function of drugs that alter modulatory neurotransmission, or to screen antipsychotic and antidepressant drugs.  However, currently in neuroscience, few optical methods exist that can detect neurotransmitters with high spatial and temporal resolution in vitro or in vivo.  Brain tissue also readily scatters visible wavelengths of light currently used to perform biological imaging, and neuronal tissue and has an abundance of biomolecules that are chemically or structurally similar and therefore hard to specifically distinguish.  Furthermore, neurotransmission relevant processes occur at challenging spatial  and temporal scales.    UC Berkeley investigators have developed polymer-functionalized carbon nanotubes for in vitro and in vivo quantification of extracellular modulatory neurotransmitter levels using optical detectors. The method uses the fluorescent optical properties of polymer-functionalized carbon nanotubes to selectively report changes in concentration of specific neurotransmitters. The scheme is novel in that the detection method applies to wide variety of specific neurotransmitters, it is an optical method and therefore gives greater spatial information, and enables the potential for imaging of one or more neurotransmitters. The optical method also produces less damage to the surrounding tissue than methods that implant electrodes or cells and allows high resolution localization with other methods of optical investigation. The invention takes advantage of favorable fluorescence properties of carbon nanotubes, such as carbon nanotube emission in the near infrared and infinite fluorescence lifetime.  The near infrared emission scatters less than shorter wavelengths, enabling greater signal recovery from deeper tissue, and allows greater compatibility with other techniques. The optical properties also enable long term potentially even chronic use. 

RNA-directed Cleavage and Modification of DNA using CasY (CRISPR-CasY)

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; 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 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 protein, CasY.  CasY 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.  CasY utilizes a guide RNA to perform double stranded cleavage of DNA. The researchers introduced CRISPR-CasY into E. coli, finding that they could block genetic material introduced into the cell.  Further research results indicated that CRISPR-CasY operates in a manner analogous to CRISPR-Cas9, but utilizing an entirely distinct protein architecture containing different catalytic domains.   CasY is also expected to function under different conditions (e.g., temperature) given the environment of the organisms that CasY was expressed in.  Similar to CRISPR Cas9, CasY enzymes are expected to have a wide variety of applications in genome editing and nucleic acid manipulation.   

RNA-directed Cleavage and Modification of DNA using CasX (CRISPR-CasX)

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; 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 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 protein, CasX, from groundwater samples. CasX 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.  CasX utilizes a tracrRNA and a guide RNA to perform double stranded cleavage of DNA. The researchers introduced CRISPR-CasX into E. coli, finding that they could block genetic material introduced into the cell.  Further research results indicated that CRISPR-CasX operates in a manner analogous to CRISPR-Cas9, but utilizing an entirely distinct protein architecture containing different catalytic domains.   CasX is also expected to function under different conditions (e.g., temperature) given the environment of the organisms that CasX was expressed in.  Similar to CRISPR Cas9, CasX enzymes are expected to have a wide variety of applications in genome editing and nucleic acid manipulation. 

Compositions and Methods for Inhibiting Stem Cell Aging

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; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin;} By 2050 the number of people in the world that will be aged 65 or older is expected to nearly triple to about 1.5 billion, representing 16% of the world’s population. One aspect of aging involves a diminished capacity to repair tissues after injury. This diminished capacity is evident in certain conditions that occur with aging, such as anemia, sarcopenia (loss of muscle mass), and osteoporosis. Deterioration of adult stem cells accounts for much of aging-associated compromised tissue maintenance. Adult stem cells mostly reside in a metabolically inactive quiescent state to preserve their integrity, but convert to a metabolically active proliferative state to replenish the tissue. The signals that trigger stem cells to exit the cell cycle and re-enter quiescence, and the signal transduction leading to the transition remain elusive however.   UC Berkeley researchers have developed methods of reducing or inhibiting or reversing stem cell aging or preventing and/or reversing tissue degeneration or injury by increasing the activity and/or the level of SIRT2 in an adult stem cell or reducing the level of a nucleotide-binding domain and leucine-rich repeat- containing-3 (NLRP3) polypeptide in an adult stem cell.  

Enzymatic Synthesis Of Cyclic Dinucleotides

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; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin;} GGDEF domain-containing enzymes are diguanylate cyclases that produce cyclic di-GMP (cdiG), a second messenger that modulates the key bacterial lifestyle transition from a motile to sessile biofilm-forming state. The ubiquity of genes encoding GGDEF proteins in bacterial genomes has established the dominance of cdiG signaling in bacteria. A subfamily of GGDEF enzymes synthesizes the asymmetric signaling molecule cyclic AMP-GMP. Hybrid CDN-producing and promiscuous substrate-binding (Hypr) GGDEF enzymes are widely distributed and found in other deltaproteobacteria and have roles that include regulation of cAG signaling.  GGDEF enzymes that produce cyclic dinucleotides are especially of interest.    UC Berkeley researcher have developed a new method of preparing and using cyclic dinucleotides (CDNs) by contacting a CDN producing-enzyme (e.g., a GGDEF enzyme) with a precursor of a CDN under conditions sufficient to convert the precursor into a CDN. This method produces a variety of non-naturally occurring, asymmetric and symmetric CDNs and can be performed in vitro or in a genetically modified host cell. Also provided are CDN compositions that find use in a variety of applications such as modulating an immune response in an individual.  

Directed Evolution Of AAV Vectors That Undergo Retrograde Axonal Transport

Brain functions such as perception, cognition, and the control of movement depend on the coordinated action of large-scale neuronal networks, which are composed of local circuit modules that are linked together by long-range connections.  Such long­ range connections are formed by specialized projection neurons that often comprise several intermingled classes, each projecting to a different downstream target within the network. Projection neurons have also been implicated in the spread of several neurodegenerative diseases. Selective targeting of projection neurons for transgene delivery is important both for gaining insights into brain function and for therapeutic intervention in neurodegenerative diseases.   Viral vectors constitute an important class of tools for introducing transgenes into specific neuronal populations, but their potential for biological investigation and gene therapy is hampered by excessive virulence.  Other viruses can infect neurons when administered directly to the nervous system, with "pseudorabies", adenoviruses and lentiviruses used most commonly in animal research. However, these viruses mediate only modest levels of transgene expression, have potential for toxicity, and are currently not easily scalable for clinical or large animal studies.  Recombinant adeno-associated viruses (rAAVs) are an effective platform for in vivo gene therapy, as they mediate high-level transgene expression, are non-toxic, and evoke minimal immune responses.  rAAVs have allowed retrograde access to projection neurons, but their natural propensity for retrograde transport is low, hampering efforts to address the role of projection neurons in circuit computations or disease progression.    UCB and HHMI researchers have produced a new rAAV variant (rAAV2-retro) that permits robust retrograde access to projection neurons with efficiency comparable to classical synthetic retrograde labeling reagents.  The rAAV2-retro gene delivery system can be used on its own or in conjunction with Cre recombinase driver lines to achieve long-term, high-level transgene expression that is sufficient for effective functional interrogation of neural circuit function, as well as for CRISPR/Cas9-mediated and other genome editing in targeted neuronal populations.  As such, this designer variant of adeno-associated virus allows for efficient mapping, monitoring, and manipulation of projection neurons.

Cas13a/C2c2 - A Dual Function Programmable RNA Endoribonuclease

Bacterial adaptive immune systems employ CRISPRs and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage. Although generally targeted to DNA substrates, the Type VI CRISPR system directs interference complexes against single-stranded RNA substrates and in Type VI CRISPR systems, the single-subunit Cas13a/C2c2 protein functions as an RNA-guided RNA endonuclease.   UC Berkeley researchers have discovered that the CRISPR-Cas13a/C2c2 has two distinct RNase activities that enable both single stranded target RNA detection and multiplexed guide-RNA processing.  These dual RNase functions were found to be chemically and mechanistically different from each other and from the CRISPR RNA processing behavior of the evolutionarily unrelated CRISPR enzyme Cpf1.  Methods for detecting the single stranded target RNA were also discovered using a Cas13a/C2c2 guide RNA and a Cas13a/C2c2 protein in a sample have a plurality of RNAs as well as methods of cleaving a precursor Cas13a/C2c2 guide RNA into two or more Cas13a/C2c2 guide RNAs.  

System and Methods to Track Single Molecules

Tracking single molecules inside cells reveals the dynamics of biological processes, including receptor trafficking, signaling and cargo transport. However, individual molecules often cannot be resolved inside cells due to their high density in the cellular environment, plus it is difficult to see spatial and temporal features, such as signal transduction events at the cell surface or on intracellular compartments, with single molecule resolution. To address these problems, researchers at the University of California, Berkeley, have developed the PhotoGate device and methods in order to control the number of fluorescent particles in a region of interest. By deploying PhotoGate and applying patterned photobleaching, they have demonstrated the tracking of single particles at surface densities two orders of magnitude higher than the single-molecule detection limit. Additional experimentation enabled the observation of ligand-induced dimerization of epidermal growth factor receptors on a live cell membrane, and also measurements of the binding and the dissociation rate of single adaptor protein from early endosomes in the crowded environment of the cytoplasm. The innovative approach enables tracking of single particles at high spatial and temporal resolution, and for mapping of molecular trajectories, as well as determining complex stoichiometry and dynamics, and drives the art towards video-rate imaging of live cells with molecular (1–5 nm) resolution.

Versatile Cas9-Mediated Integration Technology

Many advancements to the Cas9 system (both the Cas9 nuclease and the sgRNA sequence) have been made to increase and optimize its efficiency and specificity.  Since many diseases and traits in humans have a complex genetic basis, multiple genomic targets must be simultaneously edited in order for diseases to be cured or for traits to be impacted.  Thus in order for CRISPR/Cas9 to be an effective gene therapeutic technology, huge swathes of the genome must be edited simultaneously, efficiently, and accurately. To address many of these issues, UC Berkeley researchers have developed a system method to rapidly manipulate multiple loci. This system allows for either sequential (maintaining inducible Cas9 present in the genome) or simultaneous (scarless excision) manipulation of Cas9 itself and can be applied to any organism currently utilizing the CRISPR technology.  The system can also be applied conveniently to create genomic libraries, artificial genome sequences, and highly programmable strains or cell lines that can be rapidly (and repeatedly) manipulated at multiple loci with extremely high efficiency.  

Salmonella-Based Gene Delivery Vectors and their Preparation

Nucleic acid-based gene interference technologies, including ribozymes and small interfering RNAs (siRNAs), represent promising gene-targeting strategies for specific inhibition of mRNA sequences of choice. A fundamental challenge to use nucleic acid-based gene interfering approaches for gene therapy is to deliver the gene interfering agents to appropriate cells in a way that is tissue/cell specific, efficient and safe. Many of the currently used vectors are based on attenuated or modified viruses, or synthetic vectors in which complexes of DNA, proteins, and/or lipids are formed in particles, and tissue-specific vectors have been only partially obtained by using carriers that specifically target certain cell types. As such, efficient and targeted delivery of M1GS sequences to specific cell types and tissues in vivo is central to developing this technology for gene targeting applications. Invasive bacteria, such as Salmonella, possess the ability to enter and transfer genetic material to human cells, leading to the efficient expression of transferred genes. Attenuated Salmonella strains have earlier been shown to function as a carrier system for delivery of nucleic acid-based vaccines and anti-tumor transgenes. Salmonella-based vectors are low cost and easy to prepare. Furthermore, they can be administrated orally in vivo, a non-invasive delivery route with significant advantage. Thus, Salmonella may represent a promising gene delivery agent for gene therapy. Scientists at UC Berkeley have developed a novel attenuated strain of Salmonella, SL101, which exhibited high gene transfer activity and low cytotoxicity/pathogenicity while efficiently delivering ribozymes, for expression in animals. Using MCMV infection of mice as the model, they demonstrated that oral inoculation of SL101 in animals efficiently delivered RNase P-based ribozyme sequence into specific organs, leading to substantial expression of ribozyme and effective inhibition of viral infection and pathogenesis. This strategy could easily be adopted deliver other gene targeting technologies.

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