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Compositions and Methods for Treating Viral Infections

Researchers at the University of California, Davis (“UC Davis”) have developed methods for screening and targeting regions of viral genomes to identify drugs that inhibit the replication of RNA viruses.

(SD2021-085) Method for sequestering RNA binding proteins to affect their activity

The main way to reduce the activity of RBPs in cells is through gene expression knockdown (i.e. siRNAs or antisense oligonucleotides). More recently, circular RNAs have been used as a competitive inhibitor of miRNA activity by capturing the Argonaute proteins – which already occurs naturally in cells. There are also no known small molecule inhibitors of RBPs.

Gene Editing in Utero via Non-Viral, Lipid Nanoparticle Delivery of mRNA Complexes

Researchers at the University of California, Davis have developed a new method of in utero gene editing through lipid nanoparticle delivery of mRNA gene editors.

Type III CRISPR-Cas System for Robust RNA Knockdown and Imaging in Eukaryotes

Type III CRISPR-Cas systems recognize and degrade RNA molecules using an RNA-guided mechanism that occurs widely in microbes for adaptive immunity against viruses. The inventors have demonstrated that this multi-protein system can be leveraged for programmable RNA knockdown of both nuclear and cytoplasmic transcripts in mammalian cells. Using single-vector delivery of the S. thermophilus Csm complex, RNA knockdown was achieved with high efficiency (90-99%) and minimal off-targets, outperforming existing technologies of shRNA- and Cas13-mediated knockdown. Furthermore, unlike Cas13, Csm is devoid of trans-cleavage activity and thus does not induce non-specific transcriptome-wide degradation and cytotoxicity. Catalytically inactivated Csm can also be used for programmable RNA-binding, which the inventors exploit for live-cell RNA imaging. This work demonstrates the feasibility and efficacy of multi-subunit CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes.

New Therapeutic Option to Treat Bone Marrow Failure (BMF) in Patients with Dyskeratosis

The inventors have developed a genome editing therapy for bone marrow failure (BMF) in people living with dyskeratosis (DC). This technology includes two novel endogenous, isogenic models to study TINF2-DC mutations.Human embryonic stem cells (hESCs) engineered to carry the TIN2-DC T284R mutation recapitulated the short telomere phenotype observed in DC patients. Yet, telomeres in TINF2-DC hESCs did not trigger DNA damage responses at telomeres or show exacerbated telomere shortening when differentiated into telomerase-negative cells. Disruption of the mutant TINF2 allele by introducing a frameshift mutation in exon 2 restored telomere length in stem cells and the replicative potential of differentiated cells. The inventors also established in vitro and in vivo human hematopoietic stem cell (hHSC) models to assess the changes in telomere length and proliferative capacity upon the introduction of TERT and TINF2 editing. In addition, the inventors demonstrated that editing at exon 2 of TINF2 that restored telomere length in hESCs could be generated in TINF2-DC patient HSCs. These experiments nominate TINF2 as a target for: CRISPR/CAS9 to elongate telomeres in patient with TINF2 mutations,CRISPR/CAS9 to elongate telomeres with other mutations causing TBD, and chemical interventions to elongate telomeres in general.BACKGROUNDBMF is a major cause of morbidity and mortality in DC and other telomere biology disorders (TBDs). Mutations in the TINF2 gene, encoding the shelterin protein TIN2, cause telomere shortening and the inherited bone marrow failure syndrome dyskeratosis congenita (DC). A lack of suitable model systems limits the mechanistic understanding of telomere shortening in the stem cells and thus hinders the development of treatment options for bone marrow failure.   

Engineering Cas12a Genome Editors with Minimized Trans-Activity

The inventors engineered a set of LbCas12a mutants through rational design and directed evolution. The engineered mutants can function as efficient genome editors with minimized trans-activity.

Nanopore Sequencing of RNA Using Reverse Transcription

This invention demonstrates that an engineered cellular reverse transcriptase is a potent motor protein that can processively thread single-stranded RNA (ssRNA) through the MspA biological nanopore in single nucleotide steps while it is synthesizing cDNA. Notably, this represents a first-ever achievement for threading of ssRNA through the engineered Mycobacterium smegmatis porin A (MspA) nanopore in discrete steps, and also for ssRNA sequencing with the MspA nanopore. The inventors constructed the “quadromer map” for ssRNA in the MspA nanopore, which is essentially a table that can convert measured nanopore ion current to RNA sequences, using ssRNAs of known sequences. In addition, the inventors discovered that the single-molecule kinetic rates of the reverse transcriptase are affected by the presence of stable RNA secondary structures. Monitoring this biophysical behavior can be used to determine RNA structures during nanopore sequencing.  Nanopore sequencing is a powerful third generation sequencing technology that offers advantages such as ultra-long read length and direct detection of chemically modified bases. One of the key components of developing a successful nanopore sequencer is identifying potent motor proteins (such as polymerases or helicases) that can thread single-stranded (ss) DNA or ssRNA through the nanopore in discrete steps with high processivity.   

(SD2021-181) Photo-activated Control of CRISPR-Cas9 Gene Editing

RNA is one of the most important biomacromolecules in the living systems, manipulating a highly complex collection of functions which are critical to the regulation of numerous cellular pathways and processes. Being the cornerstone of biology’s central dogma, numerous approached has been developed to study and manipulate the functions of RNAs. However, compared to the study of proteins and DNAs/chromosomes, our understanding of RNA’s cellular function is significantly lacking. This is partially because of the transient nature of RNA molecule.The half-life of RNA is significantly shorter than DNA and protein. Besides, the detection of RNA suffers from low copy number as low as one copy per cell. Many creative methodologies have been developed in the past few decades to address this challenging question: how to label and manipulate cellular RNAs. Apart from non-covalent approaches, covalent RNA-modifying approaches have been challenging because of the difficulties in selectively modifying a single RNA of interest among the other RNAs in cellular conditions. Comparing to non-covalent interactions, covalent strategies provide an additional level of robustness in harsh cellular conditions.Due to the covalent linkage, the conjugated functional groups will not be disassociated from the RNA of interest in most conditions. Besides, the low-molecular weight of small-molecule (< 2 kDa) minimize the perturbation of normal RNA functions. While many covalent RNA-modifying approaches have been developed, few methods allow for the selective labeling of a single post-transcriptional RNA among the complex cellular RNA pool.

Acid Degradable Solid Lipid Nanoparticles

The inventors demonstrate that polyethylene glycol (PEG) conjugated to cholesterol via an acid degradable linkage composed of an azide-benzaldehyde acetal has the potential to allow solid lipid nanoparticles (SLNs) to be PEGylated with mole ratios up to 50%. The azide-benzaldehyde acetal, has its azide in the para position, and generates stable acetals with a t ½ of > 1000 minutes at pH 7.4. These PEG-acetals can be formulated into SLNs, and stored, and then reduced prior to biological use, to generate an amino acetal that has t ½ < 60 minutes at pH 7.4 and several minutes at pH 5.0. The ultra-PEGylated lipids were efficient at transfecting a variety of organs, including the muscle, the lung, spleen and liver and were also able to transfect the blood. Acid degradable PEG-lipids have great potential for overcoming the PEG dilemma, but have previously been challenging to develop due to the synthetic challenges associated with working with acetals and their instability at pH 7.4. (SLNs contain a PEGylated lipid, generally in the 1-5% range, which is needed to maintain SLN stability, size, and tissue diffusion, and lower toxicity. However, excessive PEGylation also results in lower cell uptake and endosomal disruption — a paradox referred to as the PEG dilemma.) The inventors anticipate numerous applications of the azide-benzaldehyde acetal linker, given its unique ability to be stable prior to reductive activation. 

High-throughput Microfluidic Research Platform for Performing Versatile Single-Cell Molecular Timed-Release Assays within Droplets

Researchers at UCI have designed a high-throughput, cost-effective microfluidic platform as a research tool for performing genomic, proteomic, single-cell, pharmacological, and agricultural studies across multiple cell types.

Multiplex Epigenetic Editing using a Split-dCas9 System

Researchers at the University of California, Davis have developed a new epigenetic editing system that overcomes packaging limitations of viral delivery systems and can be used for multiplexed epigenetic editing of a genome.

Peptides for Macromolecular Delivery

This invention includes: 1) Applying amphiphilic peptides (AP) (e.g. E5-TAT, INF7-TAT, or similar peptides) in novel scenarios for delivering CRISPR-Cas9 RNPs and performing genome editing in primary human cells of substantial clinical value (human pluripotent stem cells [HSPCs], T cells) and mouse neuronal progenitor cells in vitro or in vivo. These peptides have also been used for delivering plasmid DNA as a DNA vaccine into human cells in culture, and into mouse tissue in vivo to produce a robust immune response.  2) Novel peptide sequences (derivatives of E5-TAT or INF7-TAT) with improved properties and/or improved activity (relative to the founder peptides) in delivering cargo into target cells. The inventors have created a library of related sequences, all with distinct activity in delivering cargo to different cell types. These novel peptide sequences have been applied to the same scenarios as above, with improved outcomes compared to the parent peptides, E5-TAT or INF7-TAT, in genome editing, and may provide benefits in delivery of plasmid DNA as well. This is especially valuable when delivering to cell types that are notoriously difficult to transduce, such as HSPCs and T cells, leading to new therapeutic opportunities. Background Biological macromolecules offer great potential as therapeutics but the greatest hurdle that remains is their efficient cell entry into target cell types. Cell entry is limiting for two very promising technologies, DNA vaccines and genome editing, and this invention aims to address the unmet need. DNA vaccines would provide a rapid and inexpensive approach to vaccination for a wide range of viral pathogens, but have been hampered by poor delivery of the DNA into the target cells, resulting in an immune response not potent enough for effective vaccination. DNA vaccines involve delivering a plasmid which encodes a viral protein into the nucleus of human cells, where it can be transcribed and then expressed to be recognized by the immune system. In order to improve their efficacy, DNA vaccines have been delivered via in vivo electroporation, a painful process that requires specialized equipment and repeat dosing. Genome editing holds immense therapeutic promise for correcting the genetic mutations underlying disease, or for preventing or treating non-genetic disease. Delivery of the genome editing enzymes, such as CRISPR-Cas9, into the cytosol or nuclei of cells in need of manipulation remains the largest hurdle. Delivering CRISPR-Cas9 as a ribonucleoprotein (RNP) complex offers many advantages compared to other approaches (e.g. the use of viral vectors or lipid nanoparticles), but the RNP lacks an inherent method of cell entry. Amphiphilic peptides (AP) enable transduction of macromolecules into cells and therefore the inventors have aimed to apply APs to delivering cargo such as CRISPR-Cas9 RNPs as well as plasmid DNA into target cell types. The activity of a specific AP in delivering cargo into a the cytosol of a cell is often dependent on the specific cargo being delivered as well as the specific cell type. Therefore, the inventors have created libraries of related APs with diversity in their amino acid sequence in order to allow delivery of macromolecular cargo to a range of cell types, dependent on the specific application. A peptide sequence derived from influenza hemagluttinin sequence, HA2, has been previously described and applied as an endosomolytic peptide (ELP). When the HA2 sequence is appended with TAT, a positively charged cell penetrating peptide sequence derived from the HIV TAT protein, the fusion peptide “HA2-TAT” is able to deliver macromolecular cargo across the cell membrane and also act as an ELP to allow endosomal escape. Derivatives of the HA2-TAT sequence with changes in the amino acid sequence of the peptide, such as the peptide “E5-TAT,” has improved properties compared to HA2-TAT in solubility as well as delivering cargo into cells. The INF7-TAT peptide has similar properties, where INF7 is another glutamine-rich analog of HA2 with improved properties for endosomal escape. HA2 and its derivates (E5, INF7) with and without fusion to cell penetrating peptides (HA2-TAT, E5-TAT, INF7-TAT) have been applied to delivering macromolecular cargo such as proteins and nucleic acids into cells.

(SD2020-306) Monitoring mRNA Translation by RNA Modifications -STAMP (Surveying Targets by APOBEC-Mediated Profiling)

RNA-binding proteins (RBPs) play essential roles in gene expression and other cellular functions. Thus their identification and the understanding of their mechanisms of action and regulation is key to unraveling physiology and disease. To measure translation efficiency and different steps of ribosome recruitment, the state of the art is ribosome profiling (or Ribo‐seq) and polysome profiling which uses millions of cells, sucrose gradients, centrifugation and often requires the removal of ribosomal RNA as part of the sequencing library preparation as it contaminates more than 50% of most ribosome/polysome libraries. Also, we cannot distinguish full length isoforms here, as the ribosome‐fragments are short.

Optimized Virus-like Particles for Cas9 RNPs & Transgene/HDR Template Delivery

The inventors have developed optimized methods for using virus-like particles for the co-delivery of Cas9 ribonucleoprotein complexes and: a lentiviral genome that encodes a large transgene, such as a chimeric angtigen receptor (CAR) transgene a lentiviral genome that does not encode a sgRNA expression cassette a method for nucleofecting VLPs + homology directed repair (HDR) donor template together to enhance HDR in treated cells  

Gene Editing for Improved Plant Characteristics via Modulation of Suberin Regulators

Researchers at the University of California, Davis have identified specific genetic modifications to plants that impart a variety of advantages based on modulating the presence of suberin

Precision Graphene Nanoribbon Wires for Molecular Electronics Sensing and Switch

The inventors have developed a highly scalable multiplexed approach to increase the density of graphene nanoribbon- (GNR) based transistors. The technology forms a single device/chip (scale to 16,000 to >1,000,000 parallel transistors) on a single integrated circuit for single molecule biomolecular sensing, electrical switching, magnetic switching, and logic operations. This work relates to the synthesis and the manufacture of molecular electronic devices, more particularly sensors, switches, and complimentary metal-oxide semiconductor (CMOS) chip-based integrated circuits.Bottom-up synthesized graphene nanoribbons (GNRs) have emerged as one of the most promising materials for post-silicon integrated circuit architectures and have already demonstrated the ability to overcome many of the challenges encountered by devices based on carbon nanotubes or photolithographically patterned graphene. The new field of synthetic electronics borne out of GNRs electronic devices could enable the next generation of electronic circuits and sensors.  

Methods for Measuring Protein-DNA Interactions with Long-read DNA Sequencing

The inventors present a method to modify DNA at the site of protein-DNA interactions in intact nuclei that takes advantage of the ability for long-read DNA sequencing platforms to detect base pair modifications, such as methylation, to map the location of interactions. Current techniques for profiling protein-DNA interactions with sequencing are limited by short-read DNA sequencing technology, which precludes the mapping of protein-DNA interactions in highly repetitive regions of the genome. 

(SD2018-386) Directed Pseudouridylation of Cellular RNA Via Delivery of Crispr/Cas and esgRNA Guide Combinations

Resent strategies aimed to target and manipulate RNA in living cells mainly rely on the use of antisense oligonucleotides (ASO) or engineered RNA binding proteins (RBP). Although ASO therapies have been shown great promise in eliminating pathogenic transcripts or modulating RBP binding, they are synthetic in construction and thus cannot be encoded within DNA. This complicates potential gene therapy strategies, which would rely on regular administration of ASOs throughout the lifetime of the patient. Furthermore, they are incapable of modulating the genetic sequence of RNA. Although engineered RBPs such as PUF proteins can be designed to recognize target transcripts and fused to RNA modifying effectors to allow for specific recognition and manipulation, these constructs require extensive protein engineering for each target and may prove to be laborious and costly. 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-top:0in; mso-para-margin-right:0in; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0in; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; 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; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

Novel CRISPR-Cas Enzyme Variants and Methods of Use

CRISPR-Cas systems include Cas proteins, which are involved in acquisition, targeting and cleavage of foreign DNA or RNA, and a guide RNA(s), which includes a segment that binds Cas proteins and a segment that binds to a target nucleic acid. For example, Class 2 CRISPR-Cas systems comprise a single Cas protein bound to a guide RNA, where the Cas protein binds to and cleaves a targeted nucleic acid. The programmable nature of these systems has facilitated their use as a versatile technology for use in modification of target nucleic acid.   UC Berkeley researchers have discovered novel CRISPR-Cas proteins related to other CRISPR-Cas systems that utilize a single guide RNA (sgRNA) or a combination of a tracrRNA + guide RNA to perform RNA-directed cleavage of nucleic acids that can be applicable for DNA editing and diagnostics. The enzyme can cleave the target DNA and may be used for diagnostics by utilizing its ability to cleave single-stranded DNA in trans.  

Increased Microorganism Alcohol Tolerance Via Transformation of its pntAB Locus

Researchers at the University of California, Davis have developed microorganisms with increased alcohol tolerance by modifying the organisms’ pntAB locus through expression of one or both of its pntA/pntB genes.

Protein Inhibitor of Type VI-B CRISPR-Cas System

The inventors have discovered the first protein inhibitor of the type VI-B CRISPR-Cas system. By controlling this CRISPR system, one could possibly ameliorate the toxicity and off-target cleavage activity observed with the use of the type VI CRISPR system. Moreover, these proteins can also serve as an antidote for instances where the use of CRISPR-Cas technology poses a safety risk. Additionally, this technology can also be used for engineering genetic circuits in mammalian cells. This finding is of potential importance to many companies in the CRISPR space. 

(SD2020-376) Targeted Identification Of RNA Bases That Hydrogen Bond With Protein

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