Hybridization capture approaches allow targeted high-throughput sequencing analysis at reduced costs compared to shotgun sequencing. Hybridization capture is particularly useful in analyses of genomic data from ancient, environmental, and forensic samples, where target content is low, DNA is fragmented and multiplex PCR or other targeted approaches often fail. Hybridization capture involves the use of "bait" nucleotides that capture genomic sequences that are of particular interest for the researcher. Current bait synthesis methods require large-scale oligonucleotide chemical synthesis and/or in vitro transcription. Both RNA and DNA bait generation requires synthesizing template oligonucleotides using phosphoramidite chemistry. Microarray-based synthesis generates oligonucleotides in femtomole scales with high chemical coupling error rates. Templates synthesized at small-scale require enzymatic amplification before use in hybridization capture.The solution proposed here involves a simple and highly efficient method to generate target probes using isothermal amplification. Target sequences are circularized and then amplified by rolling circle amplification. This method generates concatemers comprising thousands of copies of the target seqeuence. Restriction digestion of the amplified product then produces probes to use in target enrichment applications. 

Researchers at the University of California, Davis have developed advancements in understanding exodermal differentiation in plant roots highlighting the role of two transcription factors in plant adaptation and survival.

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Understanding the function of RNAs requires visualizing their location and dynamics in live cells. However, direct labeling and imaging individual endogenous RNAs in living cells is still needed. UC Berkeley researchers have developed a method to directly resolve individual endogenous RNA transcripts in living cells using programmable RNA-guided and RNA-targeting CRISPR-Csm complexes coupled with a variety of crRNAs that collectively span along the transcripts of interest.  The researchers demonstrated robust labeling of MAP1B and NOTCH2 mRNAs in several cell lines. We tracked NOTCH2 and MAP1B transcript transient dynamics in living cells, captured distinct mobilities of individual transcripts in different subcellular compartments, and detected translation dependent and independent RNA motions.  

         While nuclear magnetic resonance (NMR) is one of the most universal synthetic chemistry tools for its ability to measure highly specific kinetic and structural information nondestructively/noninvasively, it is costly and low-throughput primarily due to the small sample-size volumes and expensive equipment needed for stringent magnetic field homogeneity. Conversely, zero-to-ultralow field (ZULF) NMR is an emerging alternative offering similar chemical information but relaxing field homogeneity requirements during detection. ZULF NMR has been further propelled by recent advancements in key componentry, optically pumped magnetometers (OPMs), but suffers in scope due to its low sensitivity and its susceptibility to noise. It has not been possible to detect most organic molecules without resorting to hyperpolarization or 13C enrichment using ZULF NMR.         To overcome these challenges, UC Berkeley researchers have developed a multi-channel ZULF spectrometer that greatly improves on both the sensitivity and throughput abilities of state-of-the art ZULF NMR devices. The novel spectrometer was used in the first reported detection of organic molecules in natural isotopic abundance by ZULF NMR, with sensitivity comparable to current commercial benchtop NMR spectrometers. A proof-of-concept multichannel version of the ZULF spectrometer was capable of measuring three distinct chemical samples simultaneously. The combined sensitivity and throughput distinguish the present ZULF NMR spectrometer as a novel chemical analysis tool at unprecedented scales, potentially enabling emerging fields such as robotic chemistry, as well as meeting the demands of existing fields such as chemical manufacturing, agriculture, and pharmaceutical industries.

      Quantum sensing is rapidly reshaping our ability to discern chemical processes with high sensitivity and spatial resolution. Many quantum sensors are based on nitrogen-vacancy (NV) centers in diamond, with nanodiamonds (NDs) providing a promising approach to chemical quantum sensing compared to single crystals for benefits in cost, deployability, and facile integration with the analyte. However, high-precision chemical quantum sensing suffers from large statistical errors from particle heterogeneity, fluorescence fluctuations related to particle orientation, and other unresolved challenges.      To overcome these obstacles, UC Berkeley researchers have developed a novel microfluidic chemical quantum sensing device capable of high-precision, background-free quantum sensing at high-throughput. The microfluidic device solves problems with heterogeneity while simultaneously ensuring close interaction with the analyte. The device further yields exceptional measurement stability, which has been demonstrated over >103s measurement and across ~105 droplets.  Greatly surpassing the stability seen in conventional quantum sensing experiments, these properties are also resistant to experimental variations and temperature shifts. Finally, the required ND sensor volumes are minuscule, costing only about $0.63 for an hour of analysis. 

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. Theprogrammable nature of these minimal systems has facilitated their use as a versatile technology for genome editing.  CRISPR-Cas enzymes with reduced requirements for a protospacer-adjacent motif (PAM) sequence adjacent to the target site could improve the breadth of target sites available for genome editing.  UC Berkeley researchers have developed a novel PAM-loose 12a variants, nucleic acids encoding the variant Cas12a proteins and systems using these variants that make the Cas12a-based CRISPR technology much easier to design a DNA target for carrying out genome editing in human cells. 

Worldwide, over 2.5 million people live with spinal cord injury, with over 100,000 new cases occurring annually. Spinal cord injury often causes motor dysfunction below the level of the injury. For example, thoracic and lumbar spinal cord injury can cause paraplegia and cervical spinal cord injury can cause quadriplegia. Such injury is permanent and often severe and there is no effective treatment. Various neurologic diseases also involve damaged or dysfunctional spinal cord neurons. Neural stem cell grafts have potential for treating such conditions. However, it has not been possible to obtain sufficient numbers of appropriately patterned neural stem cells, having a spinal cord positional identity, for implanted cells to survive and functionally engraft.

Aberrant splicing contributes to the etiology of many inherited diseases. Pathogenic variants impact pre-mRNA splicing through a variety of mechanisms. Most notably, variants remodel the cis-regulatory landscape of pre-mRNAs by ablation or creation of splice sites, and auxiliary splicing regulatory sequences such as exonic or intronic splicing enhancers (ESE and ISE, respectively) and splicing silencers (ESS and ISS, respectively). Splicing-sensitive variants cripple the integrity of the gene, resulting in the production of a faulty message that is either unstable or encodes an internally deleted protein. Antisense oligonucleotides (ASOs) are a promising therapeutic modality for rescuing pathogenic aberrant splicing patterns as their direct base pairing abilities make them highly customizable and specific to targets. Although challenges such as toxicity, delivery and stability represent barriers to the clinical translation of ASOs, solutions to these challenges exist, as exemplified by the recent FDA approval of multiple ASO drugs.Generally, ASO's that target splicing mutations are limited to mutations in and around splicing enhancers and exonic mutations are commonly not targeted because of the idea that the mutation causes a significant change in protein function. 

Researchers at the University of California, Davis have developed improved variants of a Heparosan synthase supporting efficient synthesis of heparosan, heparin, and heparan sulfate analogs.

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Various plasmids from Michael Rape's lab, including but not limited to:pQE-UbcH5c/pET-Ube2D3-6xHispET28-E2NpET28-UEV1ApET28a-UBE2S-6xHISpET28a-E2R1-6xHIS 

Various ubiquitin plasmids from Michael Rape lab, including but not limited to: pCS2-no his-ubiquitin wtpCS2-no his-ubiquitin all RpET30a-ubiquitin (no tag)pET30a-ubiquitin K0 (no tag) 

Various plasmid constructs and cell lines for E3 Ligase project from Julia Schaletzky lab, including but not limited to:  pET28-ubiquitin wtpET28-ubiquitin deltaGGpLentiX1hygropLentiX1 blastpLentiX1 puropLentiX1 neopCS2-6xHIS-Htt-73QpCS2-6xHIS-Htt-23QpInducer Htt-23Q-GFPpInducer Htt-73Q-GFP

Stem cells have the potential to develop into different types of cells. They are key to an organism’s development. Producing stem cell lines are important for research. Currently, avian embryonic stems cells are cultured on a layer of feeder cells. Feeder cells ensure that the stem cells survive and do not differentiate into other types of cells. However, using feeder cells can be costly and inconvenient.

Researchers at the University of California, Davis have developed a unique peptide that induces cell differentiation by inhibiting cellular protein CHD4, a promising approach to target dedifferentiated cancer cells and for cell therapy.

Volumetric additive manufacturing and vat-polymerization 3D printing methods rapidly solidify freeform objects via photopolymerization, but problematically raises the local temperature in addition to degree-of-conversion (DOC). The generated heat can critically affect the printing process as it can auto-accelerate the polymerization reaction, trigger convection flows, and cause optical aberrations. Therefore, temperature measurement alongside conversion state monitoring is crucial for devising mitigation strategies and implementing process control. Traditional infrared imaging suffers from multiple drawbacks such as limited transmission of measurement signal, material-dependent absorptions, and high background signals emitted by other objects. Consequently, a viable temperature and DOC monitoring method for volumetric 3D printing doesn’t exist.To address this opportunity, UC Berkeley researchers have developed a tomographic imaging technique that detects the spatiotemporal evolution of temperature and DOC during volumetric printing. The invention lays foundations for the development of volumetric measurement systems that uniquely resolve both temperature and DOC in volumetric printing.This novel Berkeley measurement system is envisaged as an integral tool for existing manufacturing technologies, such as computed axial lithography (CAL, Tech ID #28754), and as a new research tool for commercial biomanufacturing, general fluid dynamics, and more.

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Researchers at UC Irvine have developed an organoid culture system capable of generating three-dimensional molecular gradients. This recapitulates in vivo tissue development more accurately than current two-dimensional organoid culture systems and will allow scientists to study human-specific disease mechanisms in native tissue.

UC Berkeley researchers have developed variant Listeria bacteria that have one or more  nucleic acids that encode polypeptides required for isoprenoid synthesis through the non-mevalonate pathway, wherein the Listeria bacterium grows aerobically in the presence or absence of a functional mevalonate pathway. The Listeria strain can be used to induce enhanced activation and expansion of human gamma delta T-cells and have been shown to do so in vitro.  

Cellulose, the major component of plant biomass, is considered the most abundant biopolymer. Certain microorganisms are able to convert the monomer of cellulose, glucose, into various products useful in the production of biofuels and other methods. Cellulose is highly stable, has a high storage potential, low cost, and plentiful supply. Based on these and other properties, cellulose and enzymes capable of degrading and hydrolyzing it are useful in the sequestration, storage, and production of bioenergy.  Crystalline cellulose is composed of linear polymers of β1-4 linked glucose, held in a tightly crosslinked crystalline lattice by a high degree of intermolecular hydrogen bonding. This structure confers stability but also hinders efficient deconstruction of cellulose. Strategies for commercial depolymerization of cellulose typically combine pretreatment to disrupt the crystalline structure, followed by enzymatic hydrolysis. Disruption of the crystalline structure and chemical hydrolysis typically requires high temperatures and low pH. Enzymatic hydrolysis generally occurs under milder conditions. The degree of pretreatment required and the expense of subsequent cleanup steps are affected by properties of the enzymes used. Bacteria capable of degrading cellulose include those belonging to the genera Aquifex, Rhodothermus, Thermobifida, Anaerocellum, and Caldicellulosiruptor. A recombinant thermostable endoglucanase of Aquifex aeolicus produced in E. coli showed maximal activity at 80° C. and pH 7.0 with a half-life of 2 h at 100° C.  UC Berkeley investigators have engineered a polypeptide having cellulase activity for hydrolysis and degradation of cellulose-containing biomass.

For the growing list of "undruggable" targets that lack well defined binding pockets  a consensus is emerging that successful inhibìtors will necessarily be larger and more complex than typical small molecule drugs. The targets of existing small molecule drugs make up only a small fraction of the protein encoding genome, and it is estimated that the total "druggable" genome (accessible to inhibition by classic small molecules) represents a small fraction of the total number of potential targets. The number of therapeutic targets that have been unexploited due to poor druggability, such as transcription factors and non-coding RNAs, therefore represent a vast opportunity to make therapeutic advances in virtually every disease category. Macrocycles - in particular, cyclic peptides - have shown remarkable versatility as ligands against challenging therapeutic targets such as protein-protein interactions (PPls). Cyclization is an established method for improving potency in peptides, and cyclization can dramatically improve proteolytic stability. Importantly, the synthesis of cyclic peptides is much more modular and straightforward than the synthesis of organic molecules of similar size and complexity. Large combinatorial lìbraries of cyclic peptides, derived from methods such as DNA-encoded synthesis, phage display and mRNA-d¡splay, have yielded potent inhibitors against a variety of undruggable or challenging targets.

Researchers at the University of California, Davis have developed a self-assembling, fibrous photosensitizer that targets mitochondria in tumor cells for destruction via photodynamic therapy with enhanced localization and potency.

Tech ID 32985/Case number 2018-408 describes the generation of E. coli expressed, peptide receptive MHC-I monomers and multimers using the TAPBPR chaperone. In this case, the technology was improved based upon the surprising discovery that the TAPBPR chaperone acts catalytically on MHC-I-placeholder peptide complexes to create peptide receptive MHC-I species. 

Typically, peptide receptive MHC-I multimer reagents are prepared in bacterial (E. coli) culture. While this is efficient, it does not result in glycosylation of the MHC-I peptide fragments as is done in mammalian cells. As a result, if such reagents are produced in mammalian cells, proper glycosylation would result and the reagents would have a potentially more accurate representation of the natural T-cell target.