The present disclosure relates to compositions and methods for producing carotenoids. The present disclosure provides genetically modified cells (e.g., microbes) that comprise a transgene encoding a ferredoxin protein, and methods of making and using said genetically modified cells. The present disclosure also provides genetically modified cells (e.g., microbes) that comprise a transgene encoding a CBP or a genetic modification capable of increasing the expression of a CBP, and methods of making and using said genetically modified cells.
C12N 9/00 - Enzymes, e.g. ligases (6.)ProenzymesCompositions thereofProcesses for preparing, activating, inhibiting, separating, or purifying enzymes
C12P 23/00 - Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
Geranyl pyrophosphate (GPP) is rapidly converted to farnesyl pyrophosphate in cells, which prevents the accumulation of GPP. Increased amounts of GPP production are desired for a variety of commercial purposes. This disclosure provides methods and compositions for increasing the production of geranyl pyrophosphate in cells. This disclosure provides methods and compositions related to expression of heterologous geranyl pyrophosphate synthases as well as chimeric geranyl pyrophosphate synthases.
The present disclosure relates to compositions and methods for delivery of large DNA payloads into various loci in the genomes of a population of live cells at library scale. An advantage of the present methods and compositions is that they leverage molecular biology techniques to construct an editing vector library comprising several to many large payloads configured to integrate into several to many different loci in the genomes of cells in a population of cells in an efficient and cost-effective manner. The methods involve two rounds of cloning, with each round conducted in bulk or in "one pot", resulting in a library of editing vectors.
Methods and compositions useful for increasing the production of bacteriophage T7 RNA polymerase (T7RNAP) are provided herein. In some aspects, the methods and compositions include the use of a T7RNAP inhibitory aptamer. Novel T7RNAP promoters of varying activities are also provided.
The present disclosure provides technologies for predicting a phenotype of a microbial cell using machine learning models trained using high-content imaging data (HCI). Also provided are methods of engineering a microbial cell to possess a phenotype of interest. Example phenotypes include the production of a target compound or biomolecule of interest. The provided technologies are useful for the efficient biomanufacturing of target compounds.
The present disclosure relates to methods and compositions of matter to increase the percentage of edited cells in a cell population when employing nucleic-acid guided editing methods, as well as systems and instruments for performing these methods and using these compositions.
The present disclosure relates to synthetic biology and, in particular the bioproduction of isoprenoids using heterologous expression of 3-hydroxy-3-methylglutaryl-coenzyme-A reductase (HMGR) enzyme(s).
The present disclosure provides systems, methods, and compositions for performing iterative genomic editing of live cells with curing of editing vectors from prior rounds of editing.
The current disclosure relates to compositions and methods relating to engineered polypeptides that can encode guaiene synthases that are selective for alpha-guaiene. The engineered polypeptides can also catalyze the production of increased amounts of alpha-guaiene. The disclosure also relates to host cells including the engineered polypeptides that can be used to produce large quantities of alpha-guaiene and its derivatives. The disclosure also relates to methods of producing alpha-guaiene employing said host cells.
A61K 31/352 - Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. cannabinols, methantheline
This invention relates to compositions of matter, methods and instruments for directly recruiting repair templates to CRISPR nucleases to stimulate homology-directed repair. Molecular "tethers" are described which result in an increase in the local concentration of repair templates at the site of the double-strand break made by a nuclease, thereby enhancing the rate of homology directed repair and suppressing undesired edits.
The present disclosure provides compositions of matter, methods and instruments for nucleic acid-guided nickase/reverse transcriptase fusion enzyme editing of nucleic acids in live mammalian cells, and for tracking of editing events.
The present disclosure provides compositions of matter, methods and instruments for nucleic acid-guided nickase/reverse transcriptase fusion enzyme editing of nucleic acids in live mammalian cells, and for tracking of editing events.
The disclosure provides examples of multi-parallel processes that are used to measure strain fitness of genotypically different strains of a microorganism in one or more bioreactors and the selection of strains that have improved genotypes.
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12Q 1/04 - Determining presence or kind of microorganismUse of selective media for testing antibiotics or bacteriocidesCompositions containing a chemical indicator therefor
C12N 15/70 - Vectors or expression systems specially adapted for E. coli
C12N 15/81 - Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
This invention relates to modules and automated, integrated, end-to-end closed instruments for automated mammalian cell growth and mammalian cell transfection followed by nucleic acid-guided nuclease editing in live mammalian cells.
The present disclosure relates to automated multi-module instruments, compositions and methods for performing nucleic acid-guided nuclease editing; specifically, methods, instruments, systems, and nucleic acids synthetic cassettes that improve the efficiency of gene editing using CRISPR enzymes in diploid cells - either on both chromosomes or selectively in one chromosome without incurring loss of heterozygosity (LOH) and its often deleterious effects.
Provided are methods and compositions for detecting genome editing events at the single cell level. The methods and compositions described herein utilize sequence-based methods with combinatorial barcoding to track the identity of single cells over single or multiple genome editing events.
The present disclosure provides new RNA-guided nucleases for making rational, direct edits to nucleic acids in live cells; specifically, the present disclosure provides Type V MAD nucleases (e.g., RNA-guided nucleases or RGNs) with altered PAM preferences and/or altered activity at different temperatures or fidelity, and/or varied nuclease activities; all changes that may increase the versatility of a nucleic acid-guided nuclease for certain editing tasks.
The present disclosure provides new RNA-guided nuclease systems and engineered nickases for making rational, direct edits to nucleic acids in live cells.
The present disclosure provides engineered nucleic acid-guided nickases and optimized scaffolds for making rational, direct edits to nucleic acids in live cells.
The present disclosure provides compositions of matter, methods and instruments for nucleic acid-guided nickase/reverse transcriptase fusion editing in live cells. Editing efficiency is improved using fusion proteins (e.g., the nickase-RT fusion) that retain certain characteristics of nucleic acid-directed nucleases (e.g., the binding specificity and ability to cleave one or more DNA strands in a targeted manner) combined with reverse transcriptase activity. Editing cassettes are employed, comprising a gRNA and a repair template where the 3' end of the repair template is protected from degradation.
The present disclosure relates to automated multi-module instruments, compositions and methods for performing nucleic acid-guided nuclease editing; specifically the disclosure provides nucleic acid cassettes, plasmids, vectors, and compositions comprising the same that employ homologous recombination for genome engineering by having a CRISPR nuclease cause a specific DSB while tethered to a repair nucleic acid.
Disclosed systems and methods relate to predicting the relative representation of genomic variants in an edited cell population, based on the editing cassette design representation in an editing cassette design library used to generate the edited cell population. A library of editing cassette designs is generated, and a feature vector, or sequence embedding, is developed for each design using natural language processing techniques. The feature vector may be based upon sequence attributes and editing kinetics of each cassette design as well as attributes that describe the library context. Features may include sequence embeddings generated from a neural network, linguistic-type distances, and statistical distance summaries thereof. The feature vectors are classified using one or more machine learning models, and the classified feature vectors are used to predict the representation of each design an edited cell population.
The present disclosure relates to compositions, methods, modules and automated integrated instrumentation for multiplex delivery of "landing pad" edits into the genomes of a population of live cells. The landing pads then may be leveraged to insert very large DNA sequences into the genomes of the population of live cells.
C40B 40/08 - Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
C40B 20/04 - Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
C12N 15/113 - Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides
C12N 15/63 - Introduction of foreign genetic material using vectorsVectorsUse of hosts thereforRegulation of expression
The present disclosure provides compositions of matter, methods, modules and automated multi-module instrumentation for performing editing of live cells followed by curing of editing and engine vectors from prior rounds of editing, followed by curing of the curing vector.
The present disclosure relates to methods for performing arrayed nucleic acid-guided nuclease nickase fusion editing allowing for rapid genotypic/phenotypic correlation without sequencing.
C40B 40/02 - Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cellsLibraries contained in or displayed by vectors, e.g. plasmidsLibraries containing only microorganisms or vectors
C40B 50/06 - Biochemical methods, e.g. using enzymes or whole viable microorganisms
31.
NUCLEIC ACID-GUIDED NUCLEASE OR NICKASE FUSION EDITING OF METHYLATED NUCLEOTIDES
The present disclosure relates to compositions, methods, modules and automated, integrated instrumentation to enable nucleic acid-guided nuclease or nickase fusion editing in cells and correlating the edits to the resulting cellular nucleic acid profile. In some embodiments, methylated bases in a repair template are substituted for unmethylated bases in the cellular target genome and in some embodiments, unmethylated bases are substituted for methylated bases in the cellular target genome.
The present disclosure provides compositions, methods and modules to edit live cells and to subsequently correlate the resulting cellular nucleic acids of the edited cells to the edits.
This invention relates to compositions of matter, methods, modules and instruments for automated mammalian cell growth and mammalian cell transduction followed by nucleic acid-guided nuclease editing in live mammalian cells.
This invention relates to compositions of matter, methods, modules and automated, end-to-end closed instruments for automated mammalian cell growth, reagent bundle creation and mammalian cell transfection followed by nucleic acid-guided nuclease editing in live mammalian cells.
The present disclosure is drawn to creating cassette designs for nucleic acid- guided nuclease editing. In designing editing cassettes, a set of edit specifications must first be obtained. These edit specifications are taken together with a set of configuration parameters to start a computational pipeline that generates a collection of cassette designs. The process of designing editing cassettes involves the following exemplary steps: 1 ) creation of a set of candidate cassette designs for each unique edit specification, 2) enumeration of features describing biophysical characteristics of each candidate design, 3) providing each candidate design with a score, and 4) returning a number of scored and rank-ordered candidate cassette designs for each edit specification.
In an illustrative embodiment, the present disclosure provides methods, systems and/or instruments for the automated editing of immune cells for chimeric antigen receptor therapies.
The present disclosure provides a sphere-packing lattice electroporation device configured for use as a stand-alone unit or in an automated multi-module cell processing environment and configured to decrease cell processing time and cell survival.
The present disclosure provides compositions, automated multi-module instruments and methods to increase the percentage of edited mammalian cells in a cell population when employing nucleic-acid guided editing.
The present disclosure relates to methods and compositions that allow one to identify in vivo edited cells when employing nucleic-acid guided editing. Additionally provided are automated multi-module instruments for performing editing and selection methods and using the compositions.
The present disclosure provides new RNA-guided nucleases for making rational, direct edits to nucleic acids in live cells. The present disclosure provides mined MAD-series nucleases (e.g., RNA-guided nucleases or RGNs) with varied PAM preferences, and/or varied activity in mammalian cells. In some aspects, the MAD-series system components are delivered as sequences to be transcribed (in the case of the gRNA components) and transcribed and translated (in the case of the MAD-series nuclease), and in some aspects, the coding sequence for the MAD-series nuclease and the gRNA component sequences are on the same vector.
The present disclosure provides novel RNA-guided enzymes for making rational and direct edits to the genome of live cells. The present disclosure provides novel MAD-series nucleases with varied activity in cells from different organisms. Thus, there is provided a novel MAD-series nuclease having a codon-optimized nucleic acid sequence comprising at least 65% homology to any of SEQ ID NOs: 3-7, 11, 13, 15-22, and 24.
The present disclosure provides compositions and methods to increase the percentage of edited cells in a cell population when employing nucleic-acid guided editing, as well as automated multi-module instruments for performing these methods.
Provided are methods and compositions for preparing nucleic acid fragments for sequencing by synthesis on a flow cell. The methods and compositions described herein introduce nucleotide diversity into a sample preparation that would otherwise lack nucleotide diversity due to homogeneity of the sequencing target. This invention relates to methods and compositions of matter for optimizing sample library preparation and throughput capacity for massively parallel next-generation nucleic acid sequencing platforms.
The present disclosure provides compositions and methods to increase the percentage of edited yeast cells in a cell population when employing nucleic acid-guided editing, and automated multi-module instruments for performing these methods. Thus, there is provided in one embodiment an editing vector for nucleic acid-guided nuclease editing in yeast comprising: a promoter driving transcription of an editing cassette comprising a guide nucleic acid and a donor DNA sequence; a yeast origin of replication; a bacterial origin of replication; a promoter driving transcription of a coding sequence for a nuclease; a promoter driving transcription of a selection marker; one or more LexA DNA binding sites; and a promoter driving transcription of a LexA-linker-Rad51 fusion protein.
The present disclosure relates to various different types of variants in E. coli coding and noncoding regions leading to enhanced lysine production for, e.g., supplements and nutraceuticals. The present disclosure provides variant E.coli genes and non-coding sequences that produce enhanced amounts of lysine in culture including double and triple combinations of variant sequences.
C12N 1/21 - BacteriaCulture media therefor modified by introduction of foreign genetic material
C12N 15/00 - Mutation or genetic engineeringDNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purificationUse of hosts therefor
The present disclosure provides automated multi-module instrumentation and automated methods for performing recursive editing of live cells with curing of editing vectors from prior rounds of editing.
The present disclosure provides shuttle vectors for editing exogenous polynucleotides in heterologous live cells, as well as automated methods, modules, and multi-module cell editing instruments and systems for performing the editing methods.
THE REGENTS OF THE UNIVERSITY OF COLORADO, A BODY CORPORATE (USA)
INSCRIPTA INC. (USA)
Inventor
Batey, Robert
Iwasaki Cordero, Roman S.
Garst, Andrew
Abstract
Provided herein are single guide RNAs (sgRNAs) that comprise aptamer sequences and related compositions and methods. Also provided herein are methods of selecting inducible sgRNAs that comprise aptamer sequences.
A61K 31/7105 - Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
C12N 1/21 - BacteriaCulture media therefor modified by introduction of foreign genetic material
C12N 15/00 - Mutation or genetic engineeringDNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purificationUse of hosts therefor
The present disclosure provides a cell growth, buffer exchange, and/or cell concentration/filtration device that may be used as a stand-alone device or as a module configured to be used in an automated multi-module cell processing environment.
The present disclosure provides instruments, modules and methods for improved detection of edited cells following nucleic acid-guided nuclease genome editing. The disclosure provides improved automated instruments that perform methods—including high throughput methods—for screening cells that have been subjected to editing and identifying cells that have been properly edited.
The present disclosure provides methods, automated modules, and instruments for enrichment of live cells that have been edited by nucleic acid-guided nuclease genome editing. The disclosure provides improved methods and modules–including high throughput methods and modules–for enriching for cells that have been subjected to editing.
The present disclosure provides instruments, modules and methods for improved detection of edited cells following nucleic acid-guided nuclease genome editing. The disclosure provides improved automated instruments that perform methods — including high throughput methods — for screening cells that have been subjected to editing and identifying cells that have been properly edited.
The present disclosure provides instrumentation and automated methods for creating cell surface display libraries, where the cells of the library display engineered peptides on their cell surfaces for identification of antigens that bind to Τ-cell receptors. The engineered peptides may be putative antigens or binding regions of the T-cell receptors.
The present disclosure provides instrumentation and automated methods for creating cell surface display libraries, where the cells of the library display engineered peptides on their cell surfaces for identification of binding pairs. The engineered peptides may be displayed using various cell surface display technologies.
The present disclosure relates to methods and devices for automated control of cell growth rates where cell growth is measured in situ and the devices can be used as a stand-alone device or as a module in an automated environment, e.g., as one module in a multi-station or multi-module cell processing environment. The cell growth device comprises a temperature-controlled vial, a motor assembly to spin the vial, a spectrophotometer for measuring, e.g., OD of the cells in the vial, and a processor to accept input from a user and control the growth rate of the cells.
In an illustrative embodiment, automated multi-module cell editing instruments comprising one or more flow-through electroporation devices or modules are provided to automate genome editing in live cells.
C12M 1/42 - Apparatus for the treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic wave
C12N 13/00 - Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
C12N 15/00 - Mutation or genetic engineeringDNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purificationUse of hosts therefor
62.
AUTOMATED NUCLEIC ACID ASSEMBLY AND INTRODUCTION OF NUCLEIC ACIDS INTO CELLS
In an illustrative embodiment, automated instruments comprising one or more flow- through electroporation devices or modules are provided to automate transformation of nucleic acids in live cells.
The present disclosure provides a flow-through electroporation device configured for use as a stand-alone module or as one module in an automated multi-module processing system.
In an illustrative embodiment, automated multi-module cell editing instruments are provided to automate multiple edits into nucleic acid sequences inside one or more cells.
Disclosed herein are nucleic acid-guided nucleases, guide nucleic acids, and targetable nuclease systems, and methods of use. Disclosed herein are engineered non-naturally occurring nucleic acid-guided nucleases, guide nucleic acids, and targetable nuclease systems, and methods of use. Targetable nuclease systems can be used to edit genetic targets, including recursive genetic engineering and trackable genetic engineering methods.
Disclosed herein are engineered nucleases and nuclease systems, including chimeric nucleases and chimeric nuclease systems. Engineered and chimeric nucleases disclosed herein include nucleic acid guided nucleases. Additionally disclosed herein are methods of generating engineered nucleases and methods of using the same.
THE REGENTS OF THE UNIVERSITY OF COLORADO, A BODY CORPORATE (USA)
INSCRIPTA, INC. (USA)
Inventor
Gill, Ryan, T.
Garst, Andrew
Lipscomb, Tanya Elizabeth, Warnecke
Bassalo, Marcelo, Colika
Zeitoun, Ramsey, Ibrahim
Abstract
Provided herein are methods and composition for trackable genetic variant libraries. Further provided herein are methods and compositions for recursive engineering. Further provided herein are methods and compositions for multiplex engineering. Further provided herein are methods and compositions for enriching for editing and trackable engineered sequences and cells using nucleic acid-guided nucleases.
A01N 63/00 - Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
C12N 1/21 - BacteriaCulture media therefor modified by introduction of foreign genetic material
C12N 15/00 - Mutation or genetic engineeringDNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purificationUse of hosts therefor
patents
