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Expressing Multiple Genes From A Single Transcript In Algae And Plants

Green algae have been promoted as vehicles for the production of biofuels, pharmaceuticals, food additives, vaccines, and for toxic substance remediation, and many plants are the focus of efforts to produce drought tolerant, pest resistant, or more nutritious crops. Many of these engineering efforts rely on expression of multiple transgenes (e.g. in a multistep metabolic pathway to avoid accumulation of a toxic intermediate). It can also be useful to produce two or more proteins in a particular stoichiometry, as in a heterodimer that requires equimolar production of two polypeptides. Whether the goal is to express one transgene, or several, most efforts to transform plants and algae require cotransformation of the gene of interest with a selectable marker, such as a gene that confers resistance to a drug or herbicide, or complements an auxotrophy. Unfortunately, commonly used methods for co-transformation of algae and other plants are very inefficient. UC Berkeley investigators have developed a method for polycistronic gene expression,  and show how to achieve this using the organism's own sequences, without recourse to viral elements or other foreign elements, which is important for any technology where bioproducts are generated, since these may be used on humans (cosmetics) or in humans (food additives), especially crop technology.

Chimeric Cas9 Variants With Novel Engineered Enzymatic Activities

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

Decorating Chromatin for Precise Genome Editing Using CRISPR

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

Single Conjugative Vector for Genome Editing by RNA-guided Transposition

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

Covalent Organic Framework With Exceptional Water Sorption Properties

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

Improved Cas12a Proteins for Accurate and Efficient Genome Editing

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


The CRISPR-Cas system is now understood to confer bacteria and archaea with acquired immunity against phage and viruses. CRISPR-Cas systems consist of Cas proteins, which are involved in acquisition, targeting and cleavage of foreign DNA or RNA, and a CRISPR array, which includes direct repeats flanking short spacer sequences that guide Cas proteins to their targets.  Class 2 CRISPR-Cas are streamlined versions in which a single Cas protein bound to RNA is responsible for binding to and cleavage of a targeted sequence. The programmable nature of these minimal systems has facilitated their use as a versatile technology that is revolutionizing the field of genome manipulation.  Current CRISPR Cas technologies are based on systems from cultured bacteria, leaving untapped the vast majority of organisms that have not been isolated.  There is a need in the art for additional Class 2 CRISPR/Cas systems (e.g., Cas protein plus guide RNA combinations).     UC Berkeley researchers discovered a new type of Cas 12 protein.  Site-specific binding and/or cleavage of a target nucleic acid (e.g., genomic DNA, ds DNA, RNA, etc.) can occur at locations (e.g., target sequence of a target locus) determined by base-pairing complementarity between the Cas12 guide RNA (the guide sequence of the Cas12 guide RNA) and the target nucleic acid.  Similar to CRISPR Cas9, Cas12 enzymes are expected to have a wide variety of applications in genome editing and nucleic acid manipulation.    

Targeted Ionophore-Based Metal Supplementation

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

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.

Improved Energy Harvesting for Current-Carrying Conductors

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.

Precision Irrigation System Using Passive Mechanical Valves And Mobile Robots

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.

An Ultra-Sensitive Method for Detecting Molecules

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. 

Methods For High Signal-To-Noise Imaging Of Chromosomal Loci In Cells Using Fluorescent Cas9

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.

DNA Demethylases and uses thereof

  Normal 0 0 1 137 783 UC Berkeley 6 1 961 11.1282 0 0 0 Imprinting regulates a number of genes essential for normal development in mammals and angiosperms. In mammals such imprinted genes contribute to the control of fetal growth and development. Human diseases may also be linked to mutations in imprinted genes or aberrant regulation of their expression.. Differential DNA methylation can be established during oogenesis or spermatogenesis by de novo methyltransferases and maintained somatically by methyltransferases. The conversion of cytosine to 5'-methylcytosine in promoter associated CpG islands has been linked to changes in chromatin structure and often results in transcriptional silencing of the associated gene. Transcriptional silencing by DNA methylation has been linked to mammalian development, imprinting and X-Chromosome inactivation, suppression of parasitic DNA and numerous cancer types. This invention provides for demethylase polypeptides that excise methylated cytosines in DNA.

Dna Recombination In Eukaryotic Cells By The Bacteriophage Phic31 Recombination System

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.

Photosynthetic Hydrogen Production Using Algae

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.

Monoclonal Antibodies For Identification Of Prunus Necrotic Ringspot Virus And Related Stone Fruit Viruses

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.

Wireless Systems For Process Monitoring

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 Mutant Of The Green Alga Dunaliella Salina Accumulates Zeaxanthin, A High-value Bio-product

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

A Universal, Light-switchable Gene Promoter System

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.

Genetic Functions Required For Gene Silencing In Maize

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.

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