A cornerstone of bacterial molecular biology is the ability to genetically manipulate the microbe under study. Manipulating the genomes of bacteria is critical to many fields. Such manipulations are made by genetic engineering, which often requires new pieces of DNA to be added to the genome. It is often difficult to move genes into a recalcitrant destination organism due to surveillance systems (CRISPR, Restriction Modification) of the destination/host which degrade invading DNA . It may be commercially desirable to evade these systems in the destination organism. However, evading these systems may require significant experimental effort to design and implement.

A groundbreaking method for the efficient isolation and preservation of high-purity small extracellular vesicles (sEVs - exosomes) from biofluids using a novel EXO-PEG-TR reagent.

Efficient delivery and expression of exogenous proteins in cell populations (e.g., cells in the body) for gene therapy / gene editing applications, is an important goal in biomedicine. This can be hampered by inefficient transport of enzymes from outside the body to cells within the body. When delivering nucleic acids or proteins of interest (e.g., DNA editing enzymes), most delivery methods can only reach and enter a small subset of cells within a tissue. There is a need for compositions and methods for improved delivery of proteins of interest, and such is provided herein. UC Berkeley researchers have discovered that delivery of a molecular cargo to a target cell can be more efficiently achieved by using a cell as the delivery vehicle. This can be accomplished by delivering a nucleic acid encoding an enveloped delivery vehicle (EDV) (one that comprises a molecular cargo), to a producer cell where the producer cell produces the EDV and thereby delivers the molecular cargo to neighboring cells (referred to herein as receiver cells). Thus, there is no human intervention between delivery of a subject nucleic acid (encoding the EDV) and subsequent delivery of EDVs to target cells (receiver cells).  

Researchers from UC San Diego developed a new technology that facilitates pooling of nuclear expressed antisense RNAs (NEARs) to identify consequential RNA processing events such as alternative or constitutive RNA splicing or polyadenylation.This technology will identify a phenotype of interest and/or a group of RNA processing events (for example RNA splicing sites of interest or alternatively spliced exons), and transduce cells with a library of NEARs targeting these events. Applications include: Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4 /* 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-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman",serif;} Research tool. As screens to identify exons of phenotypic relevance in a high throughput manner.Therapeutic target identification. To identify therapeutic targets of cancer cell suppression, such as poison exons in cancer specific transcripts.Therapeutic discovery. As a therapeutic agent to identify therapeutic NEARs for splicing related disorders.  

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. 

Bento is an open-source software toolkit that uses single-molecule information to enable spatial analysis at the subcellular scale. Bento ingests molecular coordinates and segmentation boundaries to perform three analyses: defining subcellular domains, annotating localization patterns, and quantifying gene-gene colocalization. The toolkit is compatible with datasets produced by commercial and academic platforms. Bento is integrated with the open-source single-cell analysis software ecosystem.

Researchers at the University of California, Davis have identified DNA methylation biomarkers in placenta, as well as maternal and newborn blood, allowing early autism diagnosis and risk assessment.

Lipid Nanoparticles (LNPs) are a leading platform for nucleic acid delivery, widely used in therapeutics and vaccine development. However, the process of optimizing new LNP formulations has been significantly hindered by labor-intensive and costly screening methods, which require individual injections into animal models. Given the vast array of potential lipid compositions and formulation variables, these constraints severely impede the efficiency of research and development.To overcome these challenges, UC Berkeley researchers have developed a novel approach for identifying and characterizing functional nucleic acid delivery vehicles. This innovative method leverages circular RNA barcoding technology, enabling a more efficient screening process. Instead of relying on conventional cell sorting techniques, which restrict screening to specific organs and host species, this breakthrough allows direct detection of barcoded nucleic acids within circular RNAs in treated cells. By analyzing the barcodes detected, researchers can accurately determine which lipid compositions and formulations successfully delivered RNA molecules.  This technology represents a significant advancement in LNP research, offering a scalable, cost-effective solution that enhances the precision and scope of nucleic acid delivery screening.

Researchers at the University of California, Davis have developed a technology enabling hybrid crops to reproduce cloned seeds, boosting yield and stability.

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.

Professor Giulia Palermo and colleagues from the University of California, Riverside and the University of Rochester have developed high-fidelity Cas13a variants with increased sensitivity for base pair mismatches.The activation of these Cas13a variants can be inhibited with a single mismatch between guide-RNA and target-RNA, a property that can be used for the detection of SNPs associated with diseases or specific genotypic sequences.  

Researchers at the University of California, Davis have developed a novel cell segmentation technology for accurate analysis of non-spherical cells and that offers a comprehensive, high-throughput approach for analyzing the transcriptomic and metabolomic data to study complex biological processes at the single-cell level.

This technology offers a groundbreaking approach to map biomolecules in 3D space with subcellular resolution, revolutionizing our understanding of tissue organization and disease propagation.

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. 

mRNA-based cancer therapies include vaccination via mRNA delivery of tumor neoantigens, delivery of mRNA encoding for immune checkpoint and other protein therapeutics, and induced expression of anticancer surface proteins such as CAR expression in T cells. Success requires transfection of a critical number of immune cells together with appropriate immune-stimulation to effectively drive anti-tumor responses. UC Berkeley researchers have developed an adjuvant-assisted mRNA LNP delivery method that uses mRNA LNP and adjuvant to enhance delivery of nucleic acids to immune cells in vivo and stimulate immune cells. They demonstrated the use of this system to reduce mRNA reporter protein expression in the liver and enhance protein expression in the spleen in mice and also demonstrated this system can be used to genetically engineer T cells by delivering a Cre-recombinase mRNA construct- transfection and editing of approximately 4% of T cells is achieved in vivo. The immune response is superior in our system compared to current, commercial lipid nanoparticle delivery technologies.

Deoxyribonucleic acid- (DNA-) based identification in forensics is typically accomplished via genotyping allele length at a defined set of short tandem repeat (STR) loci via polymerase chain reaction (PCR). These PCR assays are robust, reliable, and inexpensive. Given the multiallelic nature of each of these loci, a small panel of STR markers can provide suitable discriminatory power for personal identification. Massively parallel sequencing (MPS) technologies and genotype array technologies invite new approaches for DNA-based identification. Application of these technologies has provided catalogs of global human genetic variation at single-nucleotide polymorphic (SNP) sites and short insertion-deletion (INDEL) sites. For example, from the 1000 Genomes Project, there is now a catalog of nearly all human SNP and INDEL variation down to 1% worldwide frequency. Genotype files, generated via MPS or genotype array, can be compared between individuals to find regions that are co-inherited or identical-by-descent (IBD). These comparisons are the basis of the relative finder functions in many direct-to-consumer genetic testing products. A special case of relative-finding is self-identification. This is a trivial comparison of genotype files as self-comparisons will be identical across all sites, minus the error rate of the assay. For many forensic samples, however, the available DNA may not be suitable for PCR-based STR amplification, genotype array analysis, or MPS to the depth required for comprehensive, accurate genotype calling. In the case of PCR, one of the most common failure modes occurs when DNA is too fragmented for amplification. For these samples, it may be possible to directly observe the degree of DNA fragmentation from the decreased amplification efficiency of larger STR amplicons from a multiplex STR amplification. In the case of severely fragmented samples, where all DNA fragments are shorter than the shortest STR amplicon length, PCR simply fails with no product.

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. 

The strategy employed by the invention is inspired by splicing factors, a category of RNA-binding protein that influence alternative splicing outcomes. These splicing factors are trans-acting, and act to enhance or silence exon inclusion by binding near or on the target exon and promoting or repressing the activity of splicing machinery. Scientifically, a highly programmable, minimally disruptive system to increase exon inclusion could allow for higher-throughput identification of functional roles of specific exons than have been previously shown.

A patent-pending platform technology designed to work in any gravity, which includes in microgravity environments, able to execute advanced molecular biology workflows; representing a paradigm shift in automation for molecular biology.

Researchers from UC San Diego developed an invention that enables protein expression to be upregulated using specific proteins and/or peptide sequences derived from SARS-CoV-2 proteins that are engineered to recognize specific mRNA transcripts by fusion to RNA-targeting modules such as CRISPR/Cas systems. They anticipate that these proteins can be fused or tethered to any engineered RNA-targeting moiety/module such as PUF/Pum, and pentatricopeptide proteins.

Delivery technologies such as lipid nanoparticles (LNP) offer significant advantages over the delivery of free RNA for various RNA therapeutic, vaccine, and basic science applications. UC Berkeley researchers developed a new class of lipid nanoparticle (LNP) which is effective in delivering various types of nuclei acids in different tissues.  The LNP was successfully tested in in-vivo mouse models and therefore poses a significant promise in the gene editing field. The lipid formulation was packaged together with CRISPR Cas9 and a gRNA targeting the endogenous Tau locus. Tau dysrregulation is a pathological feature of Alzheimers disease, thus the invention provides a means to intervene in the development of pathological states associated with Tau aggregate formation. 

Researchers at UC Berkeley have developed methods to deliver biomolecules to plant cells using new plant-derived cell penetrating peptides (CPPs). Despite the revolution in DNA editing that the last decade has brought, plant genetic engineering has not been able to benefit to the same extent. This is due to certain challenges in plant physiology that limit the delivery of exogenous protein cargos, as required in the CRISPR-Cas9 system, primarily due to the plant cell wall. In mammalian cells, for instance, cargo delivery can be accomplished using cell-penetrating peptides (CPPs) which are short peptides that facilitate the transport of cargo molecules through the plasma membrane to the cytosol. While this technology has been optimized in mammalian cells, few have studied the delivery of CPPs in plants to verify whether the cell wall is permissible to these materials. Another barrier to the use of nanotechnologies for plant biomolecule delivery is the lack of quantitative validation of successful intracellular protein delivery. The near universal dependence on confocal microscopy to validate delivery of fluorescent proxy cargoes can be inappropriate for use in plants due to various physiological plant properties, for example intrinsic autofluorescence of plant tissues. Therefore, there exists an unmet need for new materials and methods to deliver biomolecules to plant cells and to confirm the delivery of proteins of varying sizes into walled plant tissues. Stage of Research The inventors have developed methods to deliver proteins into plant cells using cell penetrating peptides which are appropriate for use with CRISPR-Cas9 technology, siRNAs, zinc-finger nucleases, TALENs, and other DNA editing methods. They have also developed a biomolecule fluorophore-based assay to accurately quantitate protein delivery to plants cells.Stage of DevelopmentResearch - in vitro 

Long read nanopore sequencing can directly sequence RNA molecules, including rRNA, and result in full-length RNA sequences. rRNA sequencing is particularly useful for identifying microbes and full-length rRNA sequencing can identify microbes with post transcriptional modifications that confer antibiotic resistance. Such post transcriptional modifications are invisible to amplification based sequencing or other sequencing techniques that require reverse transcription.Before this technology was developed, there were few if any efficient methods for preparing rRNA libraries for direct RNA sequencing, particularly for microbial identification in either a clinical or an environmental setting.   

In cells, DNA is organized by wrapping DNA strands around histone proteins, creating protein-DNA complexes called nucleosomes which comprise the basic unit of chromatin. Chromatin is associated with regions of low gene expression, as compacted DNA is inaccessible to proteins that would promote transcription. Conversely, regions in the DNA not bound by histones experience higher gene expression, as this DNA is readily available to be transcribed.  Nucleosomes are not uniformly positioned on a DNA molecule, and they change based on factors like which genes are expressed during different cellular processes. It is beneficial to understand where nucleosomes are positioned, as this can provide insight into how genes are regulated, or how factors like epigenetic modifications or chromatin structure affect this accessibility and can additionally illuminate gene expression patterns in disease for designing therapies. Nucleosome profiling is a technique used to study the positions of nucleosomes along a DNA molecule. Typically, histones are crosslinked to DNA, then the DNA is fragmented and digested leaving only regions protected by nucleosomes left for short-read sequencing. However, this fragmentation only reveals nucleosome positioning at the resolution of a few hundred base pairs, leaving the larger genomic context of these nucleosome positions to be desired. To address this, researchers at UC Santa Cruz developed Add-SEQ, a pipeline using long-read nanopore sequencing to map nucleosomes across long stretches > 10 kb of single DNA molecules.

An abasic site (i.e., an apurinic or apyrimidinic site) in a DNA or RNA strand is one in which the base is not present, but the sugar phosphate backbone remains intact. UC Santa Cruz researchers discovered that nanopore sequencers can readily detect the positions of abasic sites within a DNA strand during sequencing. This invention capitalizes on this discovery by using enzymes to generate abasic sites at places on a DNA strand that contain modified bases. The DNA strand can then be sequenced using nanopore sequencing, thereby providing a way of detecting modified bases.