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CasX Nickase Designs, Tans Cleavage Designs & Structure

Metagenomic analysis of microbial DNA from groundwater samples revealed a new protein, CasX, that prevented bacterial transformation by plasmid DNA when expressed with cognate crRNAs targeting the plasmid8. Sequence analysis of CasXrevealed no similarity to other CRISPR-Cas enzymes, except for the presence of a RuvC nuclease domain similar to that found in both Cas9 and Cas12a enzyme families as well as transposases and recombinases. The evolutionary ambiguity of CasX hinted at a distinct structure and mechanism for DNA targeting, but without reconstitution of a functional CasX enzyme it was not possible to determine its mechanism of plasmid interference.   UC Berkeley inventors found variant CasX polypeptides that induce programmable, site-specific genome repression in E. coli and genome editing in human cells, distinct from Cas9 and Cas12a, which establishes this enzyme family as a third CRISPR-Cas system for genetic manipulation.

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.

Topical Anti-proliferative Agents for Melanoma

Due to the year-to-year increase in skin cancer incidences and dramatic decrease in survival, once the melanoma has metastasized, a preventative treatment for skin cancer would be significant. Currently, the only defenses against melanoma are applying sun protection factor (SPF) regularly and protecting oneself from direct sun exposure. In a 2015 national survey conducted by the Center for Disease Control and Prevention (CDC), 34% of adults reported using SPF 15 or higher and 35% of adults reported having a sunburn in the past year.    UC Berkeley researchers have discovered active antiproliferative compounds that can be applied post-sunburn to prevent the growth and metastases of melanoma and therefore, could reduce the number of melanoma incidences per year. Based on preliminary data, they are developing a compound cocktail composed of active lead compounds to develop an anti-proliferative and cytostatic topical treatment of melanoma to slow down tumor development. 

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.  

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.  

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.   

Compositions and Methods for Regulating Ovulation

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 most widely known method for fertility control is hormonal contraception, which prevent sperm from entering the uterus. However, hormonal contraceptives present several risks associated with venous thrombosis, heart attacks, strokes, increased insulin resistance, gallbladder diseases, and liver tumors as well as a large number of side effects.  There is a need for alternatives to the currently existing contraceptives.      Polycystic ovary syndrome (PCOS) is an endocrine system disorder that affects approximatlely 10% of women of childbearing age. Almost 5 million women suffer from this condition in the United States alone.  While the exact cause of PCOS is unknown, the common symptoms include enlarged ovaries with many small cysts, inability to ovulate (hence infertility), excess hair growth, and obesity. Many women with PCOS overproduce the male sex hormone, androgens, which results in infertility. The current pharmaceutical intervention mainly includes treatment with birth control pills (steroids), however, there is no cure, because the cause of PCOS is still not known.   UC Berkeley researchers have recently discovered a novel membrane progesterone receptor that controls the ovulation. Overexpressing mice produced 3-times more eggs than control mice. The receptor acts as an enzyme that binds progesterone and produces arachidonic acid.  Arachidonic acid is converted into prostaglandins in the ovaries, and PGE2 has been shown before to trigger follicular growth and ovulation. If the enzyme levels increase, more PGE2 will be produced, and more follicles will mature. If the enzyme is suppressed, the whole ovulation process will be shut down. Since PCOS patients have much higher testosterone levels, the novel enzyme function in these patients is suppressed. So, an activator of this enzyme, which competes off testosterone, will be a better treatment or even a cure against PCOS. Compounds that modulate this enzyme’s function will promote and increase ovulation and will ultimately serve as an infertility treatment. 

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.   

Xanthene-Based Dyes For Voltage Imaging

Rapid changes in the membrane potential of excitable cells (e.g., neurons and cardiomyocytes) play a central role in defining the cellular signaling and physiological profiles of these specialized cells. Typically, the membrane potential is monitored and measured via patch clamp electrophysiology, which involves the use of a micro-electrode attached to or near the cell of interests.  Unfortunately, the use of an electrode is highly invasive, limits records to the soma of a single cell and is extremely low throughput. Researchers at the University of California, Berkeley have designed and synthesized a voltage sensitive indicator that can provide excitation and emission profiles greater than 700 nm, and as such, represents an important method for visualizing membrane potential in living cells.

Au(III) Complexes For [18F] Trifluoromethylation

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 biological properties of trifluoromethyl compounds (e.g, CF3) have led to their ubiquity in pharmaceuticals, yet their chemical properties have made their preparation a substantial challenge, necessitating innovative chemical solutions.  For example, strong, non-interacting C-F bonds lend metabolic stability while simultaneously limiting the ability of chemical transformations to forge the relevant linkages and install the CF3 unit.  When these same synthetic considerations are extended toward the synthesis of trifluoromethylated positron emission tomography (PET) tracers, the situation becomes more complex.   UC Berkeley researchers discovered an unusual alternative mechanism, in which borane abstracts fluoride from the CF3 group in a gold complex. The activated CF2 fragment can then bond to a wide variety of other carbon substituents added to the same gold center. Return of the fluoride liberates a trifluoromethylated compound from the metal. This mechanism would be useful for the introduction of radioactive fluoride substituents for potential tracers to be used for positron emission tomography applications.

Monolithically Integrated Implantable Flexible Antenna for Electrocorticography and Related Biotelemetry Devices

A sub-skin-depth (nanoscale metallization) thin film antenna is shown that is monolithically integrated with an array of neural recording electrodes on a flexible polymer substrate. The structure is intended for long-term biometric data and power transfer such as electrocorticographic neural recording in a wireless brain-machine interface system. The system includes a microfabricated thin-film electrode array and a loop antenna patterned in the same microfabrication process, on the same or on separate conductor layers designed to be bonded to an ultra-low power ASIC.

Configurations for Integrated MRI-linear Accelerators

Researchers at Stanford and University of California, Berkeley, have developed an integrated MRI-Linac hybrid system that can increase the efficacy of image-guided radiotherapy (IGRT). This system allows more aggressive treatment strategies that employ dose escalation, tighter geometric margins and sharper dose gradients which can improve clinical outcomes. This radiotherapy treatment apparatus includes a treatment beam (charged by Linac, particle, proton, or electron beam), a magnetic field disposed parallel collinear to the treatment beam, and a target that is disposed along the treatment beam. MRI is ideal for IGRT, however, there is magnetic field and RF interference between the linear accelerator and MRI scanner. The configurations of this system overcome this issue.

Small Molecule Assisted Cell Penetrating Cas9 RNP Delivery

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;} Clustered regularly interspaced short palindromic repeats (CRISPR) Cas systems provide a means for modifying genomic information and have the potential to revolutionize the treatment of genetic diseases. Although RNA-programmed Cas9 has proven to be a versatile tool for genome engineering in multiple cell types and organisms, it has been challenging to develop the therapeutics because they require the simultaneous in vivo delivery of the Cas9 protein, guide RNA and donor DNA. Compositions that can increase the efficiency of such delivery, particular in eukaryotic cells, are greatly needed.   UC Researchers have discovered that the inclusion of an agent that decreases the acidity of an endosome inside eukaryotic cells, in a genome editing composition, increased the efficiency of genome editing.  The agent was included in a composition having an RNA-guided endonuclease and an RNA-guided endonuclease and was used for gene editing.

Sensitive Detection Of Chemical Species Using A Bacterial Display Sandwich Assay

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;} Endocrine disrupting compounds are found in increasing amounts in our environment, originating from pesticides, plasticizers, and pharmaceuticals, among other sources. These compounds have been implicated in diseases such as obesity, diabetes, and cancer. The list of chemicals that disrupt normal hormone function is growing at an alarming rate, making it crucially important to find sources of contamination and identify new compounds that display this ability. However, there is currently no broad-spectrum, rapid test for these compounds, as they are difficult to monitor because of their high potency and chemical dissimilarity.   To address this, UC Berkeley researchers have developed a new detection system and method for the sensitive detection of trace compounds using electrochemical methods.  This platform is both fast and portable, and it requires no specialized skills to perform. This system enables both the detection of many detrimental compounds and signal amplification from impedance measurements due to the binding of bacteria to a modified electrode. The researchers were able to test the system finding sub-ppb levels of estradiol and ppm levels of bisphenol A in complex solutions. This approach should be broadly applicable to the detection of chemically diverse classes of compounds that bind to a single receptor.  

Sparse 3D Holographic Spatio-Temporal Focusing

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;} Several techniques are available to trigger neural activity in brain tissue on demand which are needed to study how the brain exchanges and processes information, which is useful in research and treatment applications.  The most promising solutions are all optical. Brain cells are modified with bio-compatible engineered proteins making ion-specific channels located at the neurons' cell membrane photosensitive. At this point, external triggering of action-potentials with light becomes possible.  What is needed are instruments and methods that provide specificity, improve spatial and temporal resolution, are non-invasive and bio-compatible, provide high speed and low delay, have large operating volumes.   UC Berkeley researchers have developed a new system and methods that meet the above qualities.  This new technology enables all-optical activation of individual neurons in live brain tissue and can narrowly concentrate light on individual neurons anywhere within a large 3D volume. The invention enables precise triggering of action-potentials with single neuron spatial resolution in the entire volume of interest, offering a significant improvement over existing technology.  The technology can be used as an add-on system in the optical path in a commercial microscope.  

Heterochronic Blood Exchange As A Modality To Influence Myogenesis, Neurogenesis, And Liver Regeneration

One reason for waning capabilities with advancing age is a progressive decline in organ function. Heterochronic parabiosis rejuvenates the performance of old tissues' stem cells at some expense to the young, but whether this is through shared circulatory factors or shared organ systems is unclear; and parabiosis is not a clinically adaptable approach. The old heterochronic partners have access to young organs, environmental enrichment and youthful hormones/pheromones, while the young parabiont maintains an additional aged body with deteriorating organs. In contrast to the permanent anastomosis of parabiosis, UC Berkeley researchers have used a small animal blood exchange where animals are connected and disconnected at will, removing the influence of shared organs, adaptation to being joined, etc. The effects of heterochronic blood exchange were examined with respect to all three germ layer derivatives: injured-regenerating muscle, ongoing liver cell proliferation and brain - hippocampal neurogenesis, and in the presence and absence of muscle injury.  The influence of heterochronic blood exchange on myogenesis, neurogenesis and hepatogenesis was fast, within a few days.  These findngs suggest a rapid translation of blood apheresis (FDA approved for other diseases, but not for the degenerative pathologies) for therapy to attenuate and reverse liver fibrosis and adiposity, muscle wasting and neuro-degeneration.  

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.

Voltage-Sensitive Dyes In Living Cells

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;} Comprehensively mapping and recording the electrical inputs and outputs of multiple neurons simultaneously with cellular spatial resolution and millisecond time resolution remains an outstanding challenge in the field of neurobiology. Traditionally, electrophysiology is used to directly measure membrane potential changes. While this technique yields sensitive results, it is invasive and only permits single-cell recording.  VoltageFluor dyes rely on photoinduced electron transfer to effectively report membrane potential changes in cells. This approach allows for fast, sensitive and non-invasive recording of neuronal activity in cultured mammalian neurons and in ex-vivo tissue slices. However, one major limitation of small-molecule dye imaging is the inability to target the dye to specific cells of interest.   UC Berkeley researchers have developed latent voltage sensitive dyes that require a fluorogenic activation step. This new class of VoltageFluor dyes are only weakly fluorescent until being activated in defined cell types via biological processes. In particular, the VoltageFluor dyes described herein comprise a bioreversible group that quenches the fluorescence of the VoltageFluor dye, that upon selective removal by the action of biological processes (e.g., enzymes) thereby activates the fluorescence of the VoltageFluor dye. The researchers found that the new dye facilitated the observation of spontaneous activity in rat hippocampal neurons.  

CARDIAC TISSUE MODELS AND METHODS OF USE THEREOF

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;} Tissue engineering approaches have the potential to increase the physiologic relevance of human iPS-derived cells, such as cardiomyocytes (iPS-CM). However, forming Engineered Heart Muscle (EHM) typically requires >1 million cells per tissue. Existing miniaturization strategies involve complex approaches not amenable to mass production, limiting the ability to use EHM for iPS-based disease modeling and drug screening. Micro-scale cardiospheres are easily produced, but do not facilitate assembly of elongated muscle or direct force measurements.   UC Berkeley researchers have developed a 3D filamentous fiber matrix that combines features of EHM and cardiospheres: Micro-Heart Muscle (μHM) arrays, in which elongated muscle fibers are formed in an easily fabricated template, with as few as 2,000 iPS-CM per individual tissue. Within μHM, iPS-CM exhibit uniaxial contractility and alignment, robust sarcomere assembly, and reduced variability and hypersensitivity in drug responsiveness, compared to monolayers with the same cellular composition. μHM mounted onto standard force measurement apparatus exhibited a robust Frank-Starling response to external stretch, and a dose-dependent inotropic response to the β-adrenergic agonist isoproterenol.  

Modulation Of Lymphatic Valve And Vessel Formation To Treat Diseases, Such As Trasplant Rejection

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;} Lymphatic Valve formation is associated with lymphangiogenesis, a pathological event that occurs in many diseases after inflammatory, infections, immunogenic or traumatic insults. These valves play critical roles in directing lymph flow inside the lymphatic vessels. The Lymphatic pathway is a primary mediator of immune responses, including transplant rejection. The current regimen of pharmacotherapy with corticosteroids is of limited efficacy and is fraught with serious side effects.   Researchers at the University of California, Berkeley have identified Itga-9 is critically involved in lymphatic valve formation after pathological insults, and itga-9 blockade can reduce the number of lymphatic valves formed inside the pathological lymphatic vessels. Moreover, Itga-9 interference can be used to modulate immune responses and transplant rejection. Additionally, ITga-9 can be used to improve the therapeutic effects of other anti-lymphangiogenic molecules, such as VEGFR-3. When used in combination, the formulation of both valves and lymphatic vessels are greatly suppressed and better therapeutic outcomes can be achieved for severe diseases, such as high-risk transplant rejection.  

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. 

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