Existing RNA-targeting tools for sequence-specific manipulation include anti-sense oligos (ASOs), designer PUF proteins and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas systems. However, there are significant limitations to each of the current tools. ASOs are usually not available for most RNA manipulations other than gene silencing. Designer proteins, such as PUF (Pumilio and FBF homology protein), possess low RNA recognition efficiency and it remains challenging to target RNA sequences >8-nucleotides (nt) in length. The bulky Cas protein (Cas13d: average 930 amino acids) leads to complication for transgene delivery and concerns of its immunogenicity due to its bacterial origin. Mutants of zinc finger(ZnF) proteins in ZRANB2 recognize a single-strand RNA containing a novel GGG motif with micromolar affinity, compared to the original motif GGU. These mutants serve as a foundation for RNA-binding ZnF designer protein engineering for in vivo RNA sequence-specific targeting.ZnFs are generally compact domains (~3kDa each) that have been successfully engineered for DNA recognition as modular arrays. A ZnF-based system has unique advantages, especially in a therapeutic context: (1) Broad application with the possibility to fuse with other effector domains; (2) High efficiency of RNA recognition (3 RNA bases recognized per 30-amino-acid ZnF) with a small size of protein. Only 4 ZnFs (~100 aa) is required for specific targeting in the transcriptome. (3) Humanized components without immunogenic concern.By engineering new sequence specificity of the ZRANB2 ZnF1, researchers from UC San Diego identified 13 mutants that altered their preferred RNA binding motif from GGU to GGG. They are N24R, N24H, N14D/N24R, N14D/N24H, N14R/N24R, N14R/N24H, N14H/N24R, N14H/N24H, N14Q/N24R, N14Q/N24H, N14E/N24R, N14S/N24R, N14E/N24H.

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 identified DNA methylation biomarkers in placenta, as well as maternal and newborn blood, allowing early autism diagnosis and risk assessment.

A novel bioluminescent platform for in vivo tracking and visualization of RNA dynamics without the need for excitation light.

Professor Hideaki Tsutsui and colleagues from the University of California, Riverside have developed a portable handheld device for nucleic acid extraction. With its high-speed motor, knurled lysis chamber for rapid sample lysis, and quick nucleic acid extraction using paper disks, this device can yield ready-to-use extracts in just 12 minutes, significantly reducing the time required for sample preparation. This technology is advantageous over current methods as it can be expedited without the need for cumbersome specimen collection, packaging, and submission, shortening the turnaround time.  

Researchers at the University of California, Davis have developed a method and composition for modifying genetic sequences using Adenosine deaminases that act on RNA (ADARs).

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.  

Rapid virus evolution generates proteins essential to infectivity and replication but with unknown function due to extreme sequence divergence. Using a database of 67,715 newly predicted protein structures from 4,463 eukaryotic viral species, it was found that 62% of viral proteins are structurally distinct and lack homologs in the Alphafold database. Structural comparisons suggested putative functions for >25% of unannotated viral proteins.  UC Berkeley researcher have created new single stranded DNA (ssDNA) bindingproteins and double stranded (dsDNA) binding proteins, and methods and compositions for using them, such as binding to target DNA.   

Before recombinant antibody expression plasmids can be designed, sequncing of the antibody light and heavy chain variable regions is necessary. Several other methods of sequencing antibody variable regions are available. Some involve high throughput RNA sequencing. These techniques are unavailable to many labs; they require the preparation of RNA-seq libraries, and computational analysis. As a result, the cost of performing such techniques is substantial and with sequencing cores being oversubscribed, turnaround can be as long as weeks to months. Other methods involve PCR and Sanger sequencing. However PCR amplification of variable regions results from difficulties in generating universal primers that can amplify any given variable region - particularly given the inherent low sequence identity in the 5' leader sequence of antibody light chains and heavy chains upstream of the variable regions. Sometimes degenerate primers can be used, but amplification success rate is only 80-90% due to non-specific priming and/or failure to prime at all. In addition, there is a significant risk that the variable regions of the parental myeloma line can amplify using the degenerate primers. 5' RACE (rapid amplification of 5' cDNA ends) can also be used, but mRNA degradation, cDNA purification and poly-A addition between reverse transcription and PCR, makes the technique long and difficult to perform. Non degenerate primers can be used, but each variable region requires multiple amplification attempts with different primer sets as well as sequence validation using mass spectrometry. And with both of these methods, primer derived mutations can be introduced. Mass spectrometry can be used to determine antibody variable regions, but these can result in ambiguous sequences because of isobaric residues such as isoleucine and leucine. But this method is time consuming, requires huge amounts of purified monoclonal antibody, is expensive and is inaccessible to most researchers. This technology involves a template switch reverse transcription of hybridoma RNA with at least three chain specific RT primers - one for the kappa chain, one for the lambda chain, and at least one for the heavy chain (for efficiency, this can be limited to IgG in a first pass). These are amplified in three separate PCR reactions and sequenced using Sanger sequencing.

UC Berkeley researchers have indentified and characterized a novel CRISPR Cas13 subtype that exhibits unique and advantageous features for transcriptome editing applications. At approximately half the size of the smallest known Cas13 subtype, this novel subtype is the smallest CRISPR Cas effector identified to date. The compactness of this novel Cas13 subtype facilitates its delivery into a wide array of cell types using various delivery mechanisms, significantly enhancing its utility in genomic research and therapeutic applications. The novel Cas13 subtype retains the hallmark programmable RNA-targeting capability of the Cas13 family, enabling precise and efficient editing of RNA sequences. This feature is particularly valuable in the context of transcriptome engineering, where specific alterations to RNA molecules can modulate gene expression, correct genetic errors, or modulate the function of non-coding RNAs. The discovery of this compact Cas13 subtype opens new avenues for transcriptome editing, offering potential applications in functional genomics, gene therapy, and the development of novel therapeutic strategies targeting RNA. Its ease of delivery and potent RNA-editing capabilities position this novel Cas13 subtype as a valuable tool for both basic research and clinical applications in the field of genetic engineering and precision medicine.

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.

Brief description not available

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) 

When model organisms are exposed to chemicals, resulting mutagenesis can provide insights on the chemical’s genotoxicity, which is an indicator of the chemical’s potential to cause cancer or birth defects. In fact, direct mutagenesis assays in bacteria are one of the three assays required by regulatory agencies for demonstrating the safety of potential clinical compounds. Mutagenesis assays are also used to study various DNA processes, such as replication, repair, damage tolerization, and homeostasis.

Researchers at the University of California, Davis have identified a short peptide which rapidly promotes protein degradation in cancerous enzymes and induces cell differentiation to kill lymphomas.

Researchers at the University of California, Davis have developed a virally derived homolog to increase the inflammatory response desirable in cancer immunotherapy.

Researchers at the University of California, Davis have developed a viral peptide therapeutic that targets MYC-based cancerous tumors.

Researchers at the University of California, Davis (“UC Davis”) have developed fusion proteins effective in inhibiting the replication of diverse groups of viruses that can be useful in controlling vector-borne virus transmission as well as reducing vector populations.

Presently, antiviral strategies are mostly focused on targeting viral proteins. However, the high mutation rates of RNA viruses, such as SARS-CoV-2, make the development of effective antiviral drugs very challenging. Disrupting viral-host interactions such as by targeting pro-viral, non-essential human genes will more likely prove effective against new variants or future coronavirus outbreaks.

Researchers from UC San Diego designed a new means to facilitate the enzymatic insertion of a variety of functionalized PreQ1 derivatives into a 17 nucleotide DNA hairpin which can then be appended to DNAs of interest for a variety of applications.Background: While harnessing the programmable power of nucleic acids is no new revelation for science, new innovative applications that realize this power have been crucial to scientific advancements of late. These innovative strategies often rely heavily on nucleic acid modifications.For many technical applications, precision is the key to its success, and it is necessary to have the means to carry out an efficient, site-specific, modification of the nucleic acid substrate. While a variety of site-specific enzymatic RNA modification strategies have been well established, the same is not true for DNA modifications, particularly single stranded DNA (ssDNA). Currently enzymatic modification of ssDNA is limited to 3’ insertion of modified nucleobases and the 5’ insertion of modified phosphate groups. Consequently, there is a need for higher precision methods and compositions for enzymatic modification of ssDNA.

tRNA is notoriously difficult to manipulate . Sequencing of tRNA presents various problems because of complex and tightly bound secondary structures and associated proteins. Current methods of tRNA analysis include RNA sequencing, microarray analysis and mass spectrometry. Each has limitations, however. RNA sequencing requires extensive library preparation and PCR amplification followed by reverse transcription. This results in the loss of the original RNA strand and its secondary structure. Reverse transcription is also impeded by the structural and nucleotide modifications that commonly occur in tRNA's. These so-called RTstops result in truncated cDNAs that do not reflect natural tRNA's. tRNA can be sequenced with nanopore sequencers, so long as they can be unfolded and electrically attracted to the nanopore. So a mechanism to capture tRNA molecules, unfold them, and initiate threading them into a nanopore is needed.

Classical methodologies for examining phage gene function, including UV/random mutagenesis and amber mutation, are difficult to assay efficiently on a genome-wide scale. Additionally, there are notable challenges in targeting phage genes with Cas9/12, such as epigenetic modifications, physical sequestration in the nucleus, absence of DNA genomes or intermediates in RNA phages, and efficient ligation/recombination processes. The limitation of current tools is also evident in failed attempts to apply transposon libraries in virulent phages, further underscoring the necessity for innovative approaches in phage functional genomics. UC Berkeley researchers made the surprising discovery that catalytically inactivated Cas13 (dCas13) in complex with a guide RNA can bind to and modulate activity of viral target RNAs. Viruses have evolved numerous and diverse strategies to protect their genomes from host defenses, including encoding their genomes across several Baltimore classes (e.g., dsDNA, dsRNA, ssDNA, and ssRNA), employing diverse genome modification strategies, and employing advanced genome compartmentalization strategies. These protective strategies have severely limited the applicability and effectiveness of previously existing approaches. Thus, this invention provides methods and compositions for modulating the activity of a viral target RNA.

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.

This invention overcomes a limitation of in vivo mutagenesis systems. Some methods of mutagenesis involve treatment of plasmids with mutagenic chemicals or UV light prior to transformation, but these result in biased mutation spectra. Use of error prone DNA polymerases produces a more random set of mutations, but the rate of mutagenesis rapidly declines with continuous culture. As a result, using such polymerasaes separates mutagenesis and selection into multiple steps. Mutant genes in plasmids need to be generated by the error prone polymerase, then the plasmids isolated into libraries and selected in a separate step. What is needed, then is an error prone DNA polymerase that does not result in a decline in the rate of mutagenesis in culture.