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.

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.

As opposed to short-read sequencing which requires extensive fragmentation and amplification, long-read sequencing allows researchers to sequence transcripts that span a few thousand to tens of thousands of nucleotides in length. Long-read sequencing provides several advantages over short-read sequencing due to its increased context. These benefits include enhanced confidence in mapping highly repetitive regions, the ability to determine isoform-specific expression, identification of structural variants, and characterization of epigenetic modifications. While commercial long-read technologies like PacBio and ONT require long DNA as input, currently there is no simple way to separate long and short DNA strands in a mixed pool. Standard methods are either proprietary, unreliable, low yield, highly labor intensive, or a combination of these concerns. Because of this, there is a need to develop a generalized and efficient way of isolating long DNA strands for subsequent sequencing.

Brief description not available

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.  

Currently, conventional methods of epigenome editor discovery require time and labor-intensive construct development, which is typically performed in low-throughput arrayed formats. The platform bypasses current time/labor constraints (and without reliance on construct barcodes) to facilitate the identification of an optimal gene modulator in a single experiment.

Barcodes are identifiable nucleic acid sequences that can be coupled to a target nucleic acid, either directly or indirectly. Doing so assists in analyzing the nucleic acids of interest. There are currently methods for introducing barcodes to long DNA molecules. However, long DNA molecules can be difficult to isolate. For example, long DNA molecules cannot be recovered from formalin-fixed-paraffin-embedded (FFPE) samples, but such samples are the major source of patient tumor DNA. There is a need for more efficient methods of using barcodes in haplotype phasing and other applications.   Barcoded nucleic acids on solid supports are used in a number of applications such as genome scaffolding, haplotype phasing, and single cell transcriptomics. Current methods involve the use of DNA amplification or chemical synthesis. These techniques are error prone and cost prohibitive. 

RNA-mediated adaptive immune systems in bacteria and archaea rely on Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) genomic loci and CRISPR associated (Cas) proteins that function together to provide protection from invading viruses and plasmids. Genome editing can be carried out using a CRISPR-Cas system comprising a CRISPR-Cas effector polypeptide and a guide nucleic acid, such as a guide RNA. However, unintended chromosomal abnormalities following on-target genome editing, such as chromosome loss, are potential concerns for genome editing. UC Berkeley researchers and others have developed a method to modulate the expression levels of the DNA damage response factor p53 in order to mitigate chromosomal abnormalities that occur after genome editing by nucleases like Cas9. The invention provides treatment methods by generating a modified cell and then administering the modified cell to an individual in need thereof and compositions having a CRISPR-Cas effector polypeptide, a guide nucleic acid, and an agent that increases the level of a p53 polypeptide in a mammalian cell.

Manipulation of eukaryotic genomes, particularly the integration of multi-kilobase DNAsequences, remains challenging and limits the rapidly growing fields of synthetic biology andcell engineering. Large serine recombinases (LSRs) are enzymes that recognize specific targetsequences on a DNA donor sequence and DNA target sequence to catalyze a recombinationreaction that results in the insertion of a DNA donor in a sequence-specific manner. Genomeediting can be carried out using an LSR system and a DNA donor nucleic acid, such as a plasmidor double-stranded DNA. However, in a human genome, these systems can exhibit variableefficiency and specificity.In this invention, UC Berkeley researchers and others have developed optimized compositionsto significantly increase the efficiency and specificity of LSRs to target a specific genomic locusin human cells. Via fusion to additional protein systems, this engineered composition retainsthe simplicity of a single protein for gene delivery. The invention also encompasses use in invivo or ex vivo gene therapy and the creation of modified cell lines or transgenic animals.

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. The programmable nature of these systems has facilitated their use as a versatile technology that is revolutionizing the field of genome manipulation. There is a need in the art for additional CRISPR-Cas systems with improved cleavage and manipulation under a variety of conditions and ones that are particularly thermostable under those conditions. UCB researchers created a set of efficient CRISPR-Cas9 proteins from a thermostable Cas9 from the thermophilic bacterium Geobacillus stearothermophilus (GeoCas9) through directed evolution. The gene editing activity of the evolved mutant proteins was improved by up to four orders of magnitude compared to the wild-type GeoCas9. The researchers showed that the gene editors based on the evolved GeoCas9 can be effectively assembled into lipid nanoparticles (LNP) for the rapid delivery to different cell lines in vitro as well as different organs or tissues in vivo. The LNP-based delivery strategy could also be extended to other gene editors.  

Brief description not available

Researchers at the University of California, Davis have developed a targeted epigenetic approach for the prevention and treatment CDKL5 deficiency disorder.

CRISPR-Cas systems comprise a CRISPR-associated (Cas) effector polypeptide and a guide nucleic acid. Such CRISPR-Cas systems can bind to and modify a targeted nucleic acid. The programmable nature of these CRISPR-Cas effector systems has facilitated their use as a versatile technology for use in, e.g., gene editing.   UC Berkeley researchers have discovered new CRISPR-Cas effector Cas12L/Cas Lambda/Casλ polypeptides and methods of modifying a target nucleic acid using a Cas12L/Cas Lambda polypeptide.

Human diseases that follow a dominant negative inheritance pattern present a great challenge for treatment using gene therapy methods. In such cases, a copy of an allele is inherited from each parent: one is a pathogenic allele causing a disease phenotype (e.g., by exerting a toxic, gain-of-function effect) and the other is a wild-type (non-pathogenic) allele. Allele-specific targeting is especially important when the wild-type allele is crucial to normal function, e.g., the wild-type allele encodes a protein whose function is critical. There is therefore a need for compositions and methods of allele-specific gene editing.   UC Berkeley researchers have created methods and systems for reducing the level of an RNA transcript from a target nucleic acid in an allele-specific manner. Such systems and methods can be used to treat a disease that results from or is caused by a toxic gain-of-function protein.