Researchers have developed a split-enzyme system that can detect genetic information in living cells by using luciferase linked to programmable DNA-binding domains.

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.   

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.

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. 

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.

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.

Brief description not available

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.

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  

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

Brief description not available

Brief description not available

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. 

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;}

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.

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. 

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.

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.

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 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 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 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 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.  

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/

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.