CRISPR-Cas9 is revolutionizing the field of gene editing and genome engineering. Efficient methods for delivering CRISPR-Cas9 genome editing components into target cells must be developed, both for ex vivo and in vivo applications. Current delivery strategies have drawbacks: genetically encoding Cas9 into viruses (ex. adeno-associated virus, adenovirus, retrovirus) leads to prolonged Cas9 expression in target cells, thus increasing the likelihood for off-target gene editing events. This problem can be mitigated by complexing ribonucleoprotein (RNP) Cas9 and guide RNA (gRNA) in vitro prior to administration – however, additional strategies for trafficking RNPs into target cells must additionally be employed.    To address this challenge, UC Berkeley researchers have discovered lentivirus-like particles that deliver Cas9/gRNA RNP complexes into target cells with high efficiency. This delivery strategy combines the ability of viruses to deliver cargo intracellularly with the transient nature of Cas9 RNP complexes. 

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.

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.

There are an estimated 130 million wooden poles that support overhead power lines in the US.  Extreme weather, aging, storms or sabotage can all lead to potential damage of these poles and power lines, which can leave large areas without basic necessities.  Due to this risk, it’s anticipated that power utility companies will deploy sensors and corresponding energy harvesters to better respond to potential damage of this critical electricity grid infrastructure. To address this anticipated mass deployment of sensors and harvesters, researchers at UC Berkeley have developed technology improvements to harvesting of electrical energy from energized conductors carrying alternating currents, such as those on overhead and underground power lines (as well as power-supplying conductors in offices and dwellings).  These enhanced harvesters would improve the economics of deploying sensors across a national power grid.  The Berkeley harvesters can readily provide enough power to supply wireless communication devices, energy storage batteries and capacitors, as well as sensors such as accelerometers, particulate matter measuring devices, and atmospheric sensors.

Prolonged drought in California and the Southwest has both severely reduced water allocation to farmers, and substantially increased water prices. As the drought continues, so does the pressure to increase water use efficiency and streamline water delivery practices in agriculture. The systems currently in use are insufficiently precise to satisfy the demands of high value crops such as almonds and grapes, which often require watering regimes tailored to individual plants.UC Berkeley researchers have developed a low-cost system of mechanical valves and mobile robots that will address this issue. One or more valves can be installed per plant, and periodically adjusted by the robots based on sensor data. The system provides a fine-grained control of water flow to compensate for factors that vary across the planting region.

To-date, plasmon detection methods have been utilized in the life sciences, electrochemistry, chemical vapor detection, and food safety. While passive surface plasmon resonators have lead to high-sensitivity detection in real time without further contaminating the environment with labels. Unfortunately, because these systems are passively excited, they are intrinsically limited by a loss of metal, which leads to decreased sensitivity. Researchers at the University of California, Berkeley have developed a novel method to detect distinct molecules in air under normal conditions to achieve sub-parts per billion detection limits, the lowest limit reported. This device can be used detecting a wide array of molecules including explosives or bio molecular diagnostics utilizing the first instance of active plasmon sensor, free of metal losses and operating deep below the diffraction limit for visible light.  This novel detection method has been shown to have superior performance than monitoring the wavelength shift, which is widely used in passive surface plasmon sensors. 

The ability to add a protein domain of new function is a standard molecular biology technique, and usually the domain is fused to a protein terminus. The CRISPR-associated protein Cas9 already has widespread utility for genome engineering, yet adding protein domains would increase precision and specificity. Both protein termini of Cas9, however, are close to each other and in a small defined region, which limits the effectiveness of standard fusion approaches. Therefore, insertion sites within Cas9 that will not disrupt Cas9 function are needed.Researchers at UC Berkeley have identified over 150 such sites. In proof-of-concept experiments, a PDZ protein interaction domain has been intercalated and increased functionality without decreasing Cas9 nuclease activity. In further experiments, the internal insertion sites have been used to alter Cas9 activity in an allosteric manner, effectively creating tunable Cas9.

Molecules containing aniline and aniline derivatives are common in the pharmaceutical, agrochemical, and pigment industries and numerous methods for the preparation of anilines have been reported.  Aniline derivatives containing electron-withdrawing substituents are more valuable in medicinal chemistry because anilines are prone to oxidation.  The past methods to obtain fluorinated anilines, which also mitigate oxidation, have been limited and the yields were moderate.   UC Berkeley researchers have developed a reaction for the coupling of primary fluoroalkylamines with aryl bromides and aryl chlorides and occur in the presence of functional groups that are typically not tolerated by C-N coupling reactions. The reaction yield is high and can be conducted with low catalyst loadings for most substrates.  

Cas9 is an endonuclease that binds complementary target DNA and generates site-specific breaks using two conserved nuclease domains. By inactivating both nuclease domains, dCas9 is produced, which functions as a programmable DNA binding protein. Current methods use dCas9-GFP fusions to image chromosomal loci, but have insufficient signal-to-noise ratio and often misidentify loci. UC Berkeley researchers have engineered a Cas9 variant that can be labeled with small molecule fluorescent dyes. This variant utilizes a conformational change in Cas9 to provide highly specific identification of chromosomal loci, and has been shown to work in a proof-of-principle experiment using Förster resonance energy transfer (FRET) pairs.

Lymphatic research is an explosive field of new discovery in recent years. Lymphatic dysfunction has been found in a wide array of disorders which include but are not limited to cancers and tumors, inflammation, infection, autoimmune diseases, dry eye, chemical burn, and tissue or organ transplant rejection, etc. The cornea provides an optimal site for lymphatic research due to its accessible location, transparent nature, and lymphatic-free but inducible features. Because there are no pre-existing vessels to consider in this unique tissue, it is exceptionally straightforward and accurate to assess lymphatic events (from formation to maturation and regression) in the cornea. Since lymphatic vessels are not easily visible as blood vessels, previous studies using the cornea have relied on traditional immunohistochemistry assays with dead tissues. Currently, there are no means of direct and harmless visualization of lymphatic vessels within live cornea. Investigators at University of California at Berkeley have addressed this challenge by developing the first live imaging of corneal lymphatic vessels. Lymphatic specific dye is injected into the subconjunctival space to visualize lymphatic vessels at various stages in the cornea under a fluorescence stereo, confocal, or two-photon microscope. Moreover, lymphatic vessels can be visualized in different colors to produce two, three, and four-dimensional images or live videos at a molecular level. The investigators have demonstrated a proof of principle in live mouse cornea. The technique allows time course tracking of dynamic lymphatic processes within the same tissue or subject over a short or long period of time, and can be ideally used to assess the progression of disease development and the effect of drug treatment. Live imaging of corneal lymphatic vessels allows visualization of lymphatic vessels in their natural morphology, state, and interactions with the local environment. This noninvasive method of live imaging of corneal lymphatic vessels is readily applicable to patient examination and the lymphatic dye of dextran is bio-degradable and harmless to human health.

Normal 0 0 1 95 543 UC Berkeley 4 1 666 11.1282 0 0 0 This invention relates to genes and methods to control eukaryotic mechanisms responsible for both the genesis and maintenance of heritable phenotypic variation. Paramutations represent specific types of epigenetic alterations that can be stably inherited without altering the DNA sequence. Several genes are known to be involved in regulating the paramutant state in maize plants. It is currently thought that these allelic interactions cause structural alterations to the chromatin. This invention relates to the use of the rmr1 gene for reducing or mitigating gene silencing in a transgenic maize plant and also to reduce or mitigate inbreeding depression

This invention provides methods for obtaining specific and stable integration of nucleic acids into eukaryotic cells. The method is an alternative to the widely used CRE-LOX method. The invention makes use of site-specific recombination systems that use prokaryotic recombinase polypeptides, such as the phiC31 recombinase, that can mediate recombination between the recombination sites, but not between hybrid recombination sites that are formed upon the recombination. Thus, the recombination is irreversible in the absence of additional factors. Eukaryotic cells that contain the recombinase polypeptides, or genes that encode the recombinases, are also provided. See also: MGG Molecular Genetics and Genomics. August, 2001. 265(6):1031-1038. Thomason, L. C.; Calendar, R.; Ow, D. W. Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophagevariant phiC31 site-specific recombination system. Abstract: The site-specific recombination system used by the Streptomyces bacteriophage variant phiC31 was tested in the fission yeast Schizosaccharomyces pombe. A target strain with the phage attachment site attP inserted at the leu1 locus was co-transformed with one plasmid containing the bacterial attachment site attB linked to a ura4+ marker, and a second plasmid expressing the variant phiC31 integrase gene. High-efficiency transformation to the Ura+ phenotype occurred when the integrase gene was expressed. Southern analysis revealed that the attB-ura4+ plasmid integrated into the chromosomal attP site. Sequence analysis showed that the attBXattP recombination was precise. In another approach, DNA with a ura4+ marker flanked by two attB sites in direct orientation was used to transform S. pombe cells bearing an attP duplication. The variant phiC31 integrase catalyzed two reciprocal cross-overs, resulting in a precise gene replacement. The site-specific insertions are stable, as no excision (the reverse reaction) was observed on maintenance of the integrase gene in the integrant lines. The irreversibility of the variant phiC31 site-specific recombination system sets it apart from other systems currently used in eukaryotic cells, which reverse readily. Deployment of the variant phiC31 recombination provides new opportunities for directing transgene Y and chromosome rearrangements in eukaryotic systems.

Hydrogen gas is considered to be the ideal fuel for combating environmental degradation. However, the biggest obstacle to hydrogen replacing petroleum as the world's primary source of energy is the high cost of cleanly producing this gas. The most cost-effective current method for producing H2 is to use nuclear energy -- but that has environmental issues. Likewise, using solar power is not cost-effective and using wind power is limited to a few regions. To address this challenge, researchers at the University of California, Berkeley have developed a photosynthetic method for producing H2. This patented H2 production method is based on depriving algae of sulfur which in turn inhibits oxygen flow and augments its natural H2 production. Using a bioreactor comprised of a network of sealed tubes for cultivating algae and extracting pure H2, researchers were able to produce the gas for about $0.31 per kilowatt-hour. That is much higher than natural gas-fired methods that produce H2 for about $0.05 per kilowatt-hour. However, the Berkeley team is pursuing research to address bottlenecks in this photosynthetic process which would in turn improve efficiency and reduce costs. These cost savings from the more efficient photosynthetic process along with refinements to the bioreactor design could make this algae production method cost competitive with the natural gas-fired production approach.

Prunus Necrotic Ringspot virus (PNRV) and its variants infect and have pathologic effects on a wide variety of stone fruit trees, apples, hops, and roses. This large economic impact necessitates a simple, sensitive, and highly reliable diagnostic test for these viruses in leaves, active and dormant buds, and other tissues. UC Berkeley researchers have developed 405 hybridoma lines specific for PNRV. To date, five of these have been investigated in depth. In initial tests by four laboratories, these antibodies reacted with a variety of PNRV isolates from California that commercially available antisera and monoclonal antibodies recognized more weakly or not at all.

Chemical, biochemical and agricultural processes such as fermentation, vaccine production, require close monitoring for quality control and process optimization. For some processes, production of gaseous emissions must be constantly monitored to insure worker safety or compliance with environmental regulations. Systems for many of these process monitoring applications can be very expensive and inflexible; for example where deployment requires fixed wiring for power supplies and data transmission. Systems can also be difficult to retrofit when existing facilities are used for new processing operations, or sensors must be added for monitoring new or different gaseous species. Researchers at the University of California, Berkeley have developed a wireless monitoring system for liquid processing operations. The system is designed to monitor a variety of processes, including the fermentation of wine, beer, and spirits. The system allows for rapid deployment of self-organizing sensor networks for the monitoring within production equipment (such as fermentation tanks or vats) as well as at other locations within and outside the production facility. The network can also be expanded to monitor post processing steps such as bottling or packaging. The network utilizes small, wireless sensors that are low cost and highly scalable, and the system allow for rapidly deployment into evolving liquid processing environments.

A novel mutant of of the halotolerant unicellular green alga Dunaliella salinia lacks a number of the beta-branch xanthophylls but accumulates zeaxanthin. Biochemical analysis suggests that zeaxanthin substitutes for the beta-branched xanthophylls in the mutant strain. This mutant may provide a biological source for the production of zeaxanthin, a high value bio-product. Ref.: E. Jin, & et al. 2003. Biotechnol Bioeng 81:115-124

Synopsis: This invention consists of an artificial promoter system that can be fused upstream of any desired gene, enabling reversible and light-switchable induction or repression of gene expression in any suitable host cell. New data to be filed in a provisional patent application demonstrates optimized expression conditions and a "switching off" mechanism in addition to the "switching on" mechanism.

Hollick, Jay B.; Chandler, Vicki L. Genetic factors required to maintain repression of a paramutagenic maize pl1 allele. Genetics. January, 2001. 157(1):369-378. Abstract: A genetic screen identified two novel gene functions required to maintain mitotically and meiotically heritable gene silencing associated with paramutation of the maize purple plant 1 (pl1) locus. Paramutation at pl1 leads to heritable alterations of pl1 gene regulation; the Pl-Rhoades (Pl-Rh) allele, which typically confers strong pigmentation to juvenile and adult plant structures, changes to a lower expression state termed Pl'-mahogany (Pl'). Paramutation spontaneously occurs at low frequencies in Pl-Rh homozygotes but always occurs when Pl-Rh is heterozygous with Pl'. We identified four mutations that caused increased Pl' pigment levels. Allelism tests revealed that three mutations identified two new maize loci, required to maintain repression 1 (rmr1) and rmr2 and that the other mutation represents a new allele of the previously described mediator of paramutation 1 (mop1) locus. RNA levels from Pl' are elevated in rmr mutants and genetic tests demonstrate that Pl' can heritably change back to Pl-Rh in rmr mutant individuals at variable frequencies. Pigment levels controlled by two pl1 alleles that do not participate in paramutation are unaffected rmr mutants. These results suggest that RMR functions are intimately involved in maintaining the repressed expression state of paramutant Pl' alleles. Despite strong effects on Pl' repression, rmr mutant plants have no gross developmental abnormalities even after several generations of inbreeding, implying that RMR1 and RMR2 functions are not generally required for developmental homeostasis. also see: Dorweiler, Jane E.; Carey, Charles C.; Kubo, Kenneth M.; Hollick, Jay B.; Kermicle, Jerry L.; Chandler, Vicki L. mediator of paramutation1 is required for establishment and maintenance of paramutation at multiple maize loci. Plant Cell. November, 2000. 12(11):2101-2118. Abstract: Paramutation is the directed, heritable alteration of the expression of one allele when heterozygous with another allele. Here, the isolation and characterization of a mutation affecting paramutation, mediator of paramutation1-1 (mop1-1), are described. Experiments demonstrate that the wild-type gene Mop1 is required for establishment and maintenance of the paramutant state. The mop1-1 mutation affects paramutation at the multiple loci tested but has no effect on alleles that do not participate in paramutation. The mutation does not alter the amounts of actin and ubiquitin transcripts, which suggests that the mop1 gene does not encode a global repressor. Maize plants homozygous for mop1-1 can have pleiotropic developmental defects, suggesting that mop1-1 may affect more genes than just the known paramutant ones. The mop1-1 mutation does not alter the extent of DNA methylation in rDNA and centromeric repeats. The observation that mop1 affects paramutation at multiple loci, despite major differences between these loci in their gene structure, correlations with DNA methylation, and stability of the paramutant state, suggests that a common mechanism underlies paramutation. A protein-based epigenetic model for paramutation is discussed.