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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.

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.

(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

Protein Inhibitor of Type II-A CRISPR-Cas System

The inventors have discovered three protein inhibitors of the type II-A CRISPR-Cas system that specifically inhibit Cas9 from staphylococcus aureus. This finding is of potential importance to many companies in the CRISPR space. 

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;}

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. 

Targeted Identification Of Rna Bases That Hydrogen Bond With Protein

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;} RNA binding proteins are increasingly implicated in genetic and somatic diseases.  Higher resolution methods to identify their RNA targets and how the proteins may interact with specific bases within them are needed to develop drugs that interfere with the regulation or misregulation of RBPs via their binding sites.

High-yielding Extraction of Single-Stranded Nucleic Acids with Carbon Nanotubes

PCR amplification is widely used in basic biological research and medical diagnostic tests for various infectious diseases, and is a powerful tool for nucleic acid detection. Nucleic acid extraction is an important part of the overall workflow in PCR-based viral infection test, since its function is to separate out viral nucleic acid from the many other biological components in a nasal swab-derived sample. UC Berkeley researchers have developed a method for single-stranded nucleic acid extraction from complex biofluids with DNA-wrapped carbon nanotubes. Large viral single-stranded nucleic acids can be captured by corresponding DNA-wrapped carbon nanotubes and can be concentrated for subsequent polymerase chain reaction (PCR) amplification. This method can extract nucleic acids without complicated manufacturing and experimental processes, can generate higher extraction yields than a conventional commercial PCR kit, and fits into the current PCR workflow while requiring minimal chemical reagents.  

Improved guide RNA and Protein Design for CasX-based Gene Editing Platform

The inventors have developed two new CasX gene-editing platforms (DpbCasXv2 and PlmCasXv2) through rationale structural engineering of the CasX protein and gRNA, which yield improved in vitro and in vivo behaviors. These platforms dramatically increase DNA cleavage activity and can be used as the basis for further improving CasX tools.The RNA-guided CRISPR-associated (Cas) protein CasX has been reported as a fundamentally distinct, RNA-guided platform compared to Cas9 and Cpf1. Structural studies revealed structural differences within the nucleotide-binding loops of CasX, with a compact protein size less than 1,000 amino acids, and guide RNA (gRNA) scaffold stem. These structural differences affect the active ternary complex assembly, leading to different in vivo and in vitro behaviors of these two enzymes.

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. 

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.  

Profiling Translation Rate With Ribo-Eclip

The eukaryotic ribosome is composed of 79 ribosomal protein – large (RPL) and ribosomal protein – small (RPS) subunit proteins that interweave with 4 highly structured RNAs (5S, 5.8S, 18S, and 28S rRNAs) to form the final translation-capable ribonucleoprotein. Thus, quantification of ribosome-associated RNA is highly similar to profiling of RNAs associated with other RNA binding proteins. We recently described the development of enhanced crosslinking and immunoprecipitation (eCLIP), a method to profile RNAs bound by an RNA binding protein of interest that showed thousand-fold improved recovery of protein-bound RNA [Van Nostrand et al 2016]. Van Nostrand EL, Pratt GA, Shishkin AA, Gelboin-Burkhart C, Fang MY, Sundararaman B, Blue SM, Nguyen TB, Surka C, Elkins K, et al: Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat Methods 2016, 13:508-514. https://pubmed.ncbi.nlm.nih.gov/27018577/

Expressing Multiple Genes From A Single Transcript In Algae And Plants

Green algae have been promoted as vehicles for the production of biofuels, pharmaceuticals, food additives, vaccines, and for toxic substance remediation, and many plants are the focus of efforts to produce drought tolerant, pest resistant, or more nutritious crops. Many of these engineering efforts rely on expression of multiple transgenes (e.g. in a multistep metabolic pathway to avoid accumulation of a toxic intermediate). It can also be useful to produce two or more proteins in a particular stoichiometry, as in a heterodimer that requires equimolar production of two polypeptides. Whether the goal is to express one transgene, or several, most efforts to transform plants and algae require cotransformation of the gene of interest with a selectable marker, such as a gene that confers resistance to a drug or herbicide, or complements an auxotrophy. Unfortunately, commonly used methods for co-transformation of algae and other plants are very inefficient. UC Berkeley investigators have developed a method for polycistronic gene expression,  and show how to achieve this using the organism's own sequences, without recourse to viral elements or other foreign elements, which is important for any technology where bioproducts are generated, since these may be used on humans (cosmetics) or in humans (food additives), especially crop technology.

Composition and Methods of a Nuclease Chain Reaction for Nucleic Acid Detection

This invention leverages the nuclease activity of CRISPR proteins for the direct, sensitive detection of specific nucleic acid sequences. This all-in-one detection modality includes an internal Nuclease Chain Reaction (NCR), which possesses an amplifying, feed-forward loop to generate an exponential signal upon detection of a target nucleic acid.Cas13 or Cas12 enzymes can be programmed with a guide RNA that recognizes a desired target sequence, activating a non-specific RNase or DNase activity. This can be used to release a detectable label. On its own, this approach is inherently limited in sensitivity and current methods require an amplification of genetic material before CRISPR-base detection. 

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