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ENABLING GENETIC ANALYSIS OF DIVERSE BACTERIA WITH MOBILE-CRISPRi

Researchers at UCSF, funded in part by the Chan Zuckerberg Biohub, have generated a modular and effective CRISPRi system for the genetic dissection of non-model bacteria.

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.

Synthetic Minimal Hammerhead Nuclease Ribozymes With Greatly Enhanced And Efficient Specific Cleavage Activity

The hammerhead RNA sequence within satellite RNA genomes occurs at theinterface of two monomeric segments of a linear concatamer following rolling circle replication. Although it is, in that context, a single self-cleaving strand of RNA that is capable of catalyzing only a single, albeit highly specific, cleavage reaction, the hammerhead RNA can be artificially engineered to create a true multiple-turnover ribozyme simply by separating the molecule into discrete catalytic and target strands. The latter constructs have been studied in vitro and also correspond to hammerhead ribozyme sequences that have used in targeting other RNAs

Multiple Passes of an Individual Single-Stranded Nucleic Acid through a Nanopore

A nanopore sensor can be used to sequence nucleic acid polymers by suspending a protein channel in a membrane and applying a voltage across the membrane.  When a nucleic acid polymer passes through the nanopore, it partially blocks the ionic current through the nanopore in a characteristic way unique to the sequence of the polymer. At the time of this disclosure, nanopore sequencing techniques could only provide a single read of the nucleic acid polymer. Multiple reads of the polymer could improve accuracy of nanopore sequencing. 

Salt-Tolerant Dna Polymerases

Various scientific and industrial applications exist in which it would be advantageous to use a DNA polymerase that function efficiently at high salt concentrations. In sequencing, GC compressions can be resolved by using high salt concentrations. In nanopore sequencing high salt con­centration boosts the signal to noise ratio for ionic-current ­based nanopore measurements.

TMI-seq: Tn5 Transposase Mediated Production of Complex Libraries for Short Read Sequencing

Although Next Generation Sequencing has vastly improved sequencing throughput while reducing sequencing costs, preparation of nucleic acid libraries for sequencing has become a bottleneck. In addition, it is difficult using short read next generation sequencing to assemble highly variable sequences that exceed 500 base pairs such as cDNAs derived from antibody heavy chain, antibody light chain, and T cell variable regions RNA.  

(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.

Methods Of Use Of Cas12L/CasLambda In Plants

UC researchers have discovered a novel use of proteins denoted CasLamda/Cas12L within the Type V CRISPR Cas superfamily distantly related to CasX, CasY and other published type V sequences.  These CasLamda/Cas12L proteins utilize a guide RNA to perform RNA-directed cleavage of DNA.  The researchers have developed compounds and structures for use in in editing plant cells.

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.

SageSeq for Discovering Large Deletions Produced by Genome Editing Protocols

Gene editing typically involves the use of a targeted nuclease to induce double-strand DNA breaks (DSBs) at specific genomic sites. DSBs are then repaired by one of two cellular mechanisms: homology-directed repair (HDR) which uses a DNA template to repair the DSB, while nonhomologous end joining (NHEJ) directly repairs the DSB but frequently creates an insertion or deletion mutation (indel) at the DSB site. Because HDR is usually less efficient than NHEJ, even protocols that use a DNA template to edit the region around a DSB result in a large proportion of repaired alleles containing an indel. Current methods for determining the genotypes produced by genome editing involve Polymerase Chain Reaction (PCR) amplification of DNA fragments followed by deep sequencing. These methods have limitations and do not inform the full extent of induced indel landscape. Accordingly, new and improved methods for discovering indels induced by a genome editing process is needed. The researchers discovered methods of generating a catalogue of mutations induced by a genome editing protocol that can provide an unbiased, full landscape of mutations including large indels, and/or remote indels that are distant from the targeted editing site and can identify a DNA variant un-intendedly induced by a genome editing protocol.        

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.  

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.  

2'-fluoro RNA Activators for Enhanced Activation of Csm6 in RNA Detection Assays

Csm6 constitutes a family of enzymes that are activated by cyclic oligoadenylates (cA(n)) or linear oligoadenylates with a 2´,3´-cyclic phosphate termini (A(n)>P). Cleavage of a nucleic acid sequence by an RNase to generate a linear oligoadenylate with exactly 4 or 6 A’s and the 2´,3´-cyclic phosphate terminus (A4>P or A6>P) leads to activation of Csm6/Csx1 for cleavage of a fluorescent RNA reporter. The linear A4 or A6 can be incorporated into an RNA sequence (e.g. A4-U6 or A6-U5) such that activation of Csm6 only occurs upon removal of the U-containing sequence by Cas13a, a programmable RNA-guided RNase that preferentially cleaves the phosphodiester bond that is 5’ to U’s and generates products with 2´,3´-cyclic phosphates. Csm6 is normally inactivated through self-cleavage of its activator, leading to low sensitivity when coupled with a Cas13-based RNA detection system or a Cas13-Csm6 feed-forward detection system.In this invention, the 2’-hydroxyl of the ribose in the second A in the linear A4 or the third A in the linear A6 is replaced with a 2’-fluorine (fA). This single 2’-fluoro modified RNA oligonucleotide (A-fA-AA>P or AA-fA-AAA>P) would bind and activate Csm6/Csx1 with fast kinetics and prevent degradation of the linear oligoadenylate by Csm6/Csx1. This single 2´-fluoro-modified polyA activator could be followed by any sequence to couple activation of Csm6 to a second enzyme. The purpose of this invention is to generate sustained activation of Csm6, when coupled with a Cas13 RNA detection system. In one iteration of this invention, the modified activator is followed by a linear chain of U’s, and is thus cleavable by Cas13 upon Cas13’s activation by a complementary sequence of RNA. Other nucleotides (e.g. C, A) or 2´-deoxy modifications can also be included 3´ to the first U to restrict the cleavage of Cas13a to the precise site that is required to release the single 2’-fluoro modified An>P (e.g. A-fA-AAUCCCCCC...). This activator leads to increased sensitivity and kinetics in RNA detection when coupled with Cas13. In another iteration of this invention, the modified activator is followed by a linear chain of C’s (Cn). This substrate can be acted upon by a pre-activated Csm6 (e.g. by Cas13) to produce A-fA-AA>P or AA-fA-AAA>P, which initiates a sustained feed-forward loop and prevents self-degradation of the activator by Csm6. Restricting the cleavage site of this activator by addition of chemical modifications (such as 2’-deoxy) on positions other than the cleavage site leads to a precise cut by Csm6. This activator can be combined with the previous iteration to generate even higher sensitivity and kinetics in RNA detection than the previous iteration alone. Cleavage of a fluorescent and colorimetric RNA reporter by the highly activated Csm6 in either iteration would then generate a detectable signal. In addition, nucleotides with modified bases that are not recognized by Csm6 or Cas13 may also be used in the cleavable “tail” of the activators to avoid competition with the RNA reporter or other activators in the system. Overall, the purpose of this invention is to enable elevated activation and kinetics of Csm6 when coupled with a Cas13 RNA detection system or a feed-forward reaction with Csm6 and Cas13. This could be used in low-copy detection of any type of single-stranded RNA, including viral RNA genomes, viral RNA transcripts, and cellular RNA transcripts. In addition, these activators could also be used with the related family of enzymes known as Csx1.

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.

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

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