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(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.

Stable N-acetylated analogs of Sialic Acids and Sialosides

Researchers at the University of California, Davis have constructed a library of glycans containing N-acetyl sialic acids to mimic those containing naturally occurring O-acetyl sialic acids.

2-D Polymer-Based Device for Serial X-Ray Crystallography

Researchers at the University of California, Davis have developed a single-use chip for the identification of protein crystals using X-ray based instruments.

Engineered/Variant Hyperactive CRISPR CasPhi Enzymes And Methods Of Use Thereof

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.  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. The programmable nature of these minimal 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 Class 2 CRISPR/Cas systems (e.g., Cas protein plus guide RNA combinations).     UC Berkeley researchers discovered a new type of CasPhi/12j protein.  Site-specific binding and/or cleavage of a target nucleic acid (e.g., genomic DNA, ds DNA, RNA, etc.) can occur at locations (e.g., target sequence of a target locus) determined by base-pairing complementarity between the Cas12 guide RNA (the guide sequence of the Cas12 guide RNA) and the target nucleic acid.  Similar to CRISPR Cas9, the compact Cas12 enzymes are expected to have a wide variety of applications in genome editing and nucleic acid manipulation.  

CRISPR-CAS EFFECTOR POLYPEPTIDES AND METHODS OF USE THEREOF (“Cas-VariPhi”)

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 a novel family of proteins (CasVariPhi) that utilize a guide RNA to perform RNA-directed cleavage of nucleic acids. Viral and microbial (cellular) genomes were assembled from a variety of environmental and animal microbiome sources, and variants of a novel and previously unknown Cas protein family were uncovered from the sequences decoded. 

A Point Of Care Method To Detect Covid19 Infected And Immune Patients For Pennies

The emergence of a novel coronavirus disease (COVID-19) in late 2019 has caused a worldwide health and economic crisis. Determining which members of the population are infected is key to re-opening of schools, universities, and non-essential businesses. To address this, researchers at UCI and UIC have developed an inexpensive point of care test using RNA aptamer technology for detecting COVID19 infected and immune patients that can be taken at home like a pregnancy test.

CRISPR-CAS EFFECTOR POLYPEPTIDES AND METHODS OF USE THEREOF (“Cas-Omega”)

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 a novel family of proteins (CasOmega) that utilize a guide RNA to perform RNA-directed cleavage of nucleic acids. Viral and microbial (cellular) genomes were assembled from a variety of environmental and animal microbiome sources, and variants of a novel and previously unknown Cas protein family were uncovered from the sequences decoded. 

CRISPR-CAS EFFECTOR POLYPEPTIDES AND METHODS OF USE THEREOF (“Cas-Theta”)

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 a novel family of proteins (CasTheta) that utilize a guide RNA to perform RNA-directed cleavage of nucleic acids. Viral and microbial (cellular) genomes were assembled from a variety of environmental and animal microbiome sources, and variants of a novel and previously unknown Cas protein family were uncovered from the sequences decoded. 

CRISPR-CAS EFFECTOR POLYPEPTIDES AND METHODS OF USE THEREOF (CasGamma)

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 a novel family of compact proteins (CasGamma) with a RuvC-like domain in the C-terminal end of the protein. These proteins are able to cleave nucleic acids. Viral and microbial (cellular) genomes were assembled from a variety of environmental and animal microbiome sources, and variants of a novel and previously unknown Cas protein family were uncovered from the sequences decoded. These CasGamma proteins utilize a guide RNA to perform RNA-directed cleavage of nucleic acids.  

Compositions and Methods of Isothermal Nucleic Acid Detection

An improved method for isothermal nucleic acid detection based on a loop mediated isothermal amplification (LAMP) technique that can be broadly applied for nucleic acid diagnostics.LAMP is an isothermal amplification method that amplifies DNA or RNA. This iteration of LAMP allows for the integration of any short DNA sequence, including tags, restriction enzyme sites, or promoters, into an isothermally amplified amplicon. The technique presented by the inventors allows for the insertion of sequence tags up to 35 nt into the flanking regions of the LAMP amplicon using the forward and backward inner primers (FIP and BIP), and loop primers. The inventors have demonstrated insertion of sequence fragments into the 5’ and middle regions of the FIP and BIP primers, and the 5’ region of the loop primers. In some embodiments, the sequence tag comprises a T7 RNA polymerase promoter, which is then incorporated into the LAMP amplicon (termed RT-LAMP/T7). With the addition of T7 polymerase, the amplicon can be in vitro transcribed, leading to additional amplification of the target molecule into an RNA substrate. This improves the efficiency of the amplification reaction and enables substrate conversion into different nucleic acid types.In other embodiments, the amplified RNA sequence can be detected by CRISPR enzymes, such as RNA-targeting Cas13 systems. 

XNA enzymes to Validate and Treat Genetic Diseases

Allelic proteins are often considered undruggable targets, because therapeutics that interfere with these proteins while leaving the wild-type protein unharmed are difficult to come by. Researchers at UCI have developed a xeno-nucleic enzyme (XNAzyme) that offers a solution to this problem by selectively cleaving the mRNA of mutant alleles while leaving the wild-type mRNA unharmed. This novel gene silencing technology offers an efficient, safe, and effective approach to treating genetic diseases.

COMPOSITIONS AND METHODS FOR INCREASING HOMOLOGY-DIRECTED REPAIR

Molecular self-assembly with scaffolded DNA origami offers a route for folding nucleic acid molecules in user-defined ways, to generate DNA nanostructures. DNA nanostructures have a single-stranded DNA that is folded into distinct shapes via oligonucleotides termed “staples.” Engineered nuclease systems can be used to cleave a target DNA at a specified location. Examples of engineered nuclease systems include TALENs, zinc finger nucleases, mega-nucleases, and CRISPR-Cas systems. Introduction of a break in a nucleic acid (e.g. genome) can facilitate the introduction of a donor nucleic acid.    UC Berkeley researchers have discovered compositions comprising a gene-editing polypeptide, a single-stranded donor DNA, and one or more staple oligonucleotides which can be used for gene editing. 

COMPOSITIONS AND METHODS FOR IDENTIFYING HOST CELL TARGET PROTEINS FOR TREATING RNA VIRUS INFECTIONS

Viral infection is a multistep process involving complex interplay between viral life cycle and host immunity. One defense mechanism that hosts use to protect cells against the virus are nucleic-acid-mediated surveillance systems, such as RNA interference-driven gene silencing and CRISPR-Cas mediated gene editing. Another important stage for host cells to combat virus replication is translational regulation, which is particular important for the life cycle of RNA viruses, such as Hepatitis C virus and Coronavirus.  While efforts to characterize structural features of viral RNA have led to a better understanding of translational regulation, no systematical approaches to identify important host genes for controlling viral translation have been developed and little is known about how to regulate host-virus translational interaction to prevent and treat infections caused by RNA viruses.   UC Berkeley researchers have developed a high-throughput platform using CRISPR-based target interrogation to identify new therapeutics targets or repurposed drug targets for blocking viral RNA translation.  The new kits can also be used to identify important domains within target proteins that are required for regulating (viral RNA translation) and can inform drug design and development for treating RNA viruses.

DNA Methylation Measurement For Mammals Based On Conserved Loci

UCLA researchers in the Departments of Human Genetics and Biological Chemistry have developed a new approach for measuring DNA methylation levels in mammals based on short and highly conserved nucleotide sequences.  This method facilitates the development of chip for measuring DNA methylation that can be used for cross-species comparisons and used for building universal epigenetic aging clocks (age estimators) that apply to all mammals.

4D-seq: Single Cell RNA-sequencing with in situ Spatiotemporal Information

To develop a novel imaging-based single cell RNA-sequencing (scRNA-Seq) platform that allows capturing of spatiotemporal information and cellular behavior of the sequenced cells within tissue.

In Vitro Reconstituted Plant Virus Capsids For Delivering Rna Genes To Mammalian Cells

UCLA researchers in the Department of Chemistry & Biochemistry have developed a method for using in vitro reconstituted plant virus-derived vectors to package and deliver RNA genes for targeted delivery of vaccines, MRI contrast agents, and therapeutic proteins in RNA form.

DARTS: Deep Learning Augmented RNA-seq Analysis of Transcript Splicing

Researchers led by Yi Xing have developed a novel deep learning algorithm to detect alternative splicing patterns in RNA-seq data

CRISPR-CAS EFFECTOR POLYPEPTIDES AND METHODS OF USE THEREOF

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.  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. The programmable nature of these minimal systems has facilitated their use as a versatile technology that is revolutionizing the field of genome manipulation.  Current CRISPR Cas technologies are based on systems from cultured bacteria, leaving untapped the vast majority of organisms that have not been isolated.  There is a need in the art for additional Class 2 CRISPR/Cas systems (e.g., Cas protein plus guide RNA combinations).     UC Berkeley researchers discovered a new type of Cas 12 protein.  Site-specific binding and/or cleavage of a target nucleic acid (e.g., genomic DNA, ds DNA, RNA, etc.) can occur at locations (e.g., target sequence of a target locus) determined by base-pairing complementarity between the Cas12 guide RNA (the guide sequence of the Cas12 guide RNA) and the target nucleic acid.  Similar to CRISPR Cas9, Cas12 enzymes are expected to have a wide variety of applications in genome editing and nucleic acid manipulation.    

Sustained Intracellular RNA Delivery and Expression

UCLA researchers in the Department of Chemistry and Biochemistry have developed a novel method for high protein expression levels, in situ, involving RNA-based therapeutics.

Catalytic MicroRNA Antagonists

UCLA researchers in the Department of Biological Chemistry have developed a novel approach for miRNA inhibition.

Simultaneous Detection Of Protein Isoforms And Nucleic Acids From Low Starting Cell Numbers

Embryo-specific nucleic acid modifications, including retrotransposon activity-derived genomic modifications and alternative splicing of mRNA, is crucial for the development of mammalian embryos. However, determining if all genomic modifications and mRNA isoforms translate to protein variations remain intriguing questions due to difficulty in measuring protein isoforms and nucleic acids from small starting cell numbers.    UC Researchers have developed a system for performing dual nucleic acid and protein isoform measurements on low starting cell numbers equivalent to the number of blastomeres composing early embryonic development stages (morula and blastocysts).  The system integrates fractionation polyacrylamide gel electrophoresis (fPAGE) with off-chip analysis of nucleic acids in the nuclei. An additional method can be used to remove nuclei for off-chip analysis. The system can measure expression of protein isoforms from the cytoplasmic fraction of 1-100 cells while achieving analysis of either DNA or mRNA retained in the nuclei. The researchers have demonstrated signal from immunoprobed protein correlates strongly with protein expression prior to lysis in TurboGFP-expressing cells and that mRNA levels correlate with protein abundance in TurboGFP-expressing cells.

Lentivirus-like Particle Delivery of CRISPR-Cas9 & Guide RNA for Gene Editing

CRISPR-Cas9 is revolutionizing the field of gene editing and genome engineering. Efficient methods for delivering CRISPR-Cas9 genome editing components into target cells must be developed, both for ex vivo and in vivo applications. Current delivery strategies have drawbacks: genetically encoding Cas9 into viruses (ex. adeno-associated virus, adenovirus, retrovirus) leads to prolonged Cas9 expression in target cells, thus increasing the likelihood for off-target gene editing events. This problem can be mitigated by complexing ribonucleoprotein (RNP) Cas9 and guide RNA (gRNA) in vitro prior to administration – however, additional strategies for trafficking RNPs into target cells must additionally be employed.    To address this challenge, UC Berkeley researchers have discovered lentivirus-like particles that deliver Cas9/gRNA RNP complexes into target cells with high efficiency. This delivery strategy combines the ability of viruses to deliver cargo intracellularly with the transient nature of Cas9 RNP complexes. 

Rapid, Sensitive Detection of Nucleic Acid Sequences in Environmental Samples

UCLA Researchers at the California NanoSystems Institute have developed a methodology that permits PCR-based detection of nucleic acid sequences in soil that does not require the isolation of DNA.

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