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
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.
This disclosure provides compositions and methods useful for editing a target nucleic acid molecule. This disclosure provides MAD2019-H848A variant polypeptides, reverse transcriptases, and fusion proteins and methods of using MAD2019-H848A variant polypeptides, reverse transcriptases, and fusion proteins to edit nucleic acid molecules both in vivo and in vitro.
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 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 present disclosure relates to various different types of mutations or modifications in Saccharomyces cerevisiae coding and noncoding regions leading to enhanced cellobiohydrolase I production for, e.g., supplements and nutraceuticals.
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.
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.
40 - Treatment of materials; recycling, air and water treatment,
42 - Scientific, technological and industrial services, research and design
Goods & Services
Custom manufacture of microorganisms for use in manufacturing of chemicals, small molecules, pharmaceuticals, nutraceuticals, proteins and food additives Research and development in the field of biotechnology
12.
MODULES AND INSTRUMENTS FOR AUTOMATED NUCLEIC ACID-GUIDED NUCLEASE EDITING IN MAMMALIAN CELLS USING MICROCARRIERS
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 provides engineered nucleic acid-guided nickases and optimized scaffolds for making rational, direct edits to nucleic acids in live cells.
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.
C12N 15/11 - DNA or RNA fragmentsModified forms thereof
C12Q 1/6865 - Promoter-based amplification, e.g. nucleic acid sequence-based amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
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
15.
METHODS AND COMPOSITIONS OF CO-EXPRESSION OF T7RNA POLYMERASE AND INHIBITORY RNA APTAMERS
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.
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.
C12N 15/88 - Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using liposome vesicle
C12N 15/90 - Stable introduction of foreign DNA into chromosome
The present disclosure relates to synthetic biology and, in particular, the expression of heterologous proteins in a microbial cell, and engineered enzymes for achieving the same.
The present disclosure provides compositions of matter, methods, systems, and instruments for improved nucleic acid-guided nuclease editing in live cells, wherein the live cells are shifted into a growth-arrested state for editing.
C12N 1/38 - Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factorsStimulation of growth by removal of a chemical compound
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 provides compositions of matter, methods and instruments for nucleic acid-guided nickase/reverse transcriptase fusion enzyme editing of nucleic acids in live mammalian cells.
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 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 provides systems, methods, and compositions for performing iterative genomic editing of live cells with curing of editing vectors from prior rounds of editing.
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 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.
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
Compositions of matter, methods, modules, and automated instruments may relate to synthesizing a library including an editing cassette including a different gRNA and donor DNA pair, amplifying the editing cassette in a partition separate from other editing cassettes in the library, adding nuclease to the partition, and adding lipofectamine to the editing cassette and nuclease to form a lipofectamine/nucleic acid/nuclease complex. A microcarrier coated in extracellular matrix or a cell adhesion molecule coating may be added to the lipofectamine/nucleic acid/nuclease complex. Cell growth material, the microcarrier, and mammalian cells may be transferred to a growth module in an automated closed cell editing instrument via a liquid handling system. The mammalian cells may be allowed to seed on the microcarrier. Conditions may be provided for the mammalian cells to take-up and be edited by a payload associated with the lipofectamine/nucleic acid/nuclease complex. The mammalian cells may be detached from the microcarrier.
C12N 15/11 - DNA or RNA fragmentsModified forms thereof
C12N 15/88 - Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using liposome vesicle
C12N 15/90 - Stable introduction of foreign DNA into chromosome
B01L 3/00 - Containers or dishes for laboratory use, e.g. laboratory glasswareDroppers
B01L 7/00 - Heating or cooling apparatusHeat insulating devices
C12M 1/32 - Inoculator or sampler multiple field or continuous type
C12M 3/06 - Tissue, human, animal or plant cell, or virus culture apparatus with filtration, ultrafiltration, inverse osmosis or dialysis means
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.
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, 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 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.
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.
01 - Chemical and biological materials for industrial, scientific and agricultural use
Goods & Services
Genetically-modified or biologically-modified microbes for use in the production of chemicals, small molecules, pharmaceuticals, nutraceuticals, proteins, and food additives.
The present disclosure relates to methods and compositions for nuclease-mediated plasmid integration into the genome of a population of live cells, as well as automated multi-module instruments for performing these methods and using these compositions.
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
The present disclosure relates to methods, compositions, and automated multi-module cell processing instruments for modulation of gene utilizing nuclease-mediated systems, and in particular, inactive (“dead”) nuclease-mediated CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) systems.
The present disclosure relates to cell populations and systems for detection of compounds in an environment. Specifically the disclosure relates to methods for generating reproducible genome-wide edited populations of microbes that display novel, defined, and reproducible phenotypes when exposed to one or more chemicals. In some applications, such phenotypes are read out by barcode amplicon and compared against population fingerprints. In other applications digital information is stored in such populations of microorganisms. The digital information can be retrieved from the microorganisms with Boolean logic.
C12Q 1/689 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
G16B 20/20 - Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
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.
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.
C40B 30/04 - Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
C40B 40/10 - Libraries containing peptides or polypeptides, or derivatives thereof
C07K 16/28 - Immunoglobulins, e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
C07K 14/74 - Major histocompatibility complex [MHC]
C40B 70/00 - Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or barcodes
C12N 15/81 - Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
C12N 15/85 - Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
C12N 15/90 - Stable introduction of foreign DNA into chromosome
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
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
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
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 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.
Nucleic acid-guided nuclease editing in mammalian cells may include passaging mammalian cells, in an automated closed cell editing instrument, into smaller aggregates when the aggregates exceed 50-300 microns in size. A library of viral particles may be delivered to the mammalian cells at a multiplicity of infection such that each mammalian cell receives one or no viral particle. The library may include viral vectors with an editing cassette including a pair of gRNA coding sequence and donor DNA. Conditions may be provided to allow a viral vector of the viral vectors to integrate into the mammalian cells. Enriching for mammalian cells may be done with an integrated viral vector. A nucleic acid-guided nuclease or nuclease fusion or a coding sequence for a nucleic acid-guided nuclease or nuclease fusion may be delivered to the enriched mammalian cells and conditions may be provided to allow editing in the mammalian cells.
C12N 15/63 - Introduction of foreign genetic material using vectorsVectorsUse of hosts thereforRegulation of expression
C12N 5/00 - Undifferentiated human, animal or plant cells, e.g. cell linesTissuesCultivation or maintenance thereofCulture media therefor
C12N 15/79 - Vectors or expression systems specially adapted for eukaryotic hosts
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12M 1/34 - Measuring or testing with condition measuring or sensing means, e.g. colony counters
C12N 15/11 - DNA or RNA fragmentsModified forms thereof
C12N 15/88 - Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using liposome vesicle
C12N 15/90 - Stable introduction of foreign DNA into chromosome
B01L 3/00 - Containers or dishes for laboratory use, e.g. laboratory glasswareDroppers
B01L 7/00 - Heating or cooling apparatusHeat insulating devices
C12M 1/32 - Inoculator or sampler multiple field or continuous type
C12M 3/06 - Tissue, human, animal or plant cell, or virus culture apparatus with filtration, ultrafiltration, inverse osmosis or dialysis means
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.
The present disclosure provides multiplexed engineered cells, automated multi-module instruments and methods of producing biofuel producing cells for enhanced production of biofuels. This platform empowers users to design genetic variant libraries of insertions, swaps, and/or deletions, that can be intentionally or randomly positioned across the entire genome to generate engineered cell populations with improved properties for several common biofuel applications including, but not limited to, improved tolerance to biomass inhibitors, increased thermo-tolerance, increased ethanol production and/or tolerance, expanded carbon utilization abilities, and increased utilization of heterologous proteins or pathways.
The present disclosure provides automated modules and instruments for improved detection of nuclease genome editing of live cells. The disclosure provides improved modules—including high throughput modules—for screening cells that have been subjected to editing and identifying and selecting cells that have been properly edited.
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
C12M 3/00 - Tissue, human, animal or plant cell, or virus culture apparatus
C12M 1/42 - Apparatus for the treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic wave
C12N 15/11 - DNA or RNA fragmentsModified forms thereof
C12N 15/90 - Stable introduction of foreign DNA into chromosome
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.
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 T-cell receptors. The engineered peptides may be putative antigens or binding regions of the T-cell receptors.
C40B 50/06 - Biochemical methods, e.g. using enzymes or whole viable microorganisms
C12N 15/90 - Stable introduction of foreign DNA into chromosome
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C40B 40/10 - Libraries containing peptides or polypeptides, or derivatives thereof
C12N 15/85 - Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
C07K 16/28 - Immunoglobulins, e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
C40B 70/00 - Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or barcodes
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
C07K 14/74 - Major histocompatibility complex [MHC]
C12N 15/81 - Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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
C40B 30/04 - Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
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 engineered nucleic acid-guided nickases and optimized scaffolds for making rational, direct edits to nucleic acids in live cells.
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 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 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 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.
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.
C12M 3/00 - Tissue, human, animal or plant cell, or virus culture apparatus
C12M 1/00 - Apparatus for enzymology or microbiology
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
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
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 T-cell receptors. The engineered peptides may be putative antigens or binding regions of the T-cell receptors.
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
C40B 30/04 - Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
C40B 40/10 - Libraries containing peptides or polypeptides, or derivatives thereof
C07K 16/28 - Immunoglobulins, e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
C07K 14/74 - Major histocompatibility complex [MHC]
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12N 15/81 - Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
C12N 15/85 - Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
C12N 15/90 - Stable introduction of foreign DNA into chromosome
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
C40B 70/00 - Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or barcodes
66.
gRNA STABILIZATION IN NUCLEIC ACID-GUIDED NICKASE EDITING
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 provides automated modules and instruments for improved detection of nuclease genome editing of live cells. The disclosure provides improved modules—including high throughput modules—for screening cells that have been subjected to editing and identifying and selecting cells that have been properly edited.
C12M 3/00 - Tissue, human, animal or plant cell, or virus culture apparatus
B01L 3/00 - Containers or dishes for laboratory use, e.g. laboratory glasswareDroppers
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
C12M 1/42 - Apparatus for the treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic wave
C12N 15/11 - DNA or RNA fragmentsModified forms thereof
C12N 15/90 - Stable introduction of foreign DNA into chromosome
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 relates to methods for increasing observed editing rates in the surviving bacteria cells. The compositions and methods presented herein in combination lead to a phenomenon of “edit or die.” Although less cells survive plating and editing, a large percentage of cells that do survive are multiple editors. In one experiment it was found that if a cell survives transformation, plating, and editing, 75% of the surviving cells are multiple editors; that is, 75% of the surviving cells were simultaneously edited with edits at two or more different locations within the bacterial genome.
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.
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 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. Specifically, the disclosure relates to methods, compositions, modules and automated multi-module cell processing instruments that increase the efficiency of nucleic acid-guided editing in a cell population using a nucleic acid nuclease (i.e., an RNA-guided nuclease or “RGN”)/single-strand binding protein (“SSB”) fusion system. The system leverages a single-strand binding protein (SSP) and single-strand DNA annealing protein (“SSAP”) interactions to drive enhanced recruitment.
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. The present compositions and methods entail viral delivery of an editing cassette to live mammalian cells such that the editing cassettes edit the cells and the edited cells continue to grow, preferably using a fully-automated end-to-end instrument to process the cells without human intervention to enhance cell processing uniformity and to maintain the integrity of the cell culture.
C12N 5/00 - Undifferentiated human, animal or plant cells, e.g. cell linesTissuesCultivation or maintenance thereofCulture media therefor
C12N 15/63 - Introduction of foreign genetic material using vectorsVectorsUse of hosts thereforRegulation of expression
C12N 15/85 - Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12M 1/34 - Measuring or testing with condition measuring or sensing means, e.g. colony counters
C12N 15/11 - DNA or RNA fragmentsModified forms thereof
C12N 15/88 - Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using liposome vesicle
C12N 15/90 - Stable introduction of foreign DNA into chromosome
B01L 3/00 - Containers or dishes for laboratory use, e.g. laboratory glasswareDroppers
B01L 7/00 - Heating or cooling apparatusHeat insulating devices
C12M 1/32 - Inoculator or sampler multiple field or continuous type
C12M 3/06 - Tissue, human, animal or plant cell, or virus culture apparatus with filtration, ultrafiltration, inverse osmosis or dialysis means
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 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 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.
C40B 50/06 - Biochemical methods, e.g. using enzymes or whole viable microorganisms
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
C07K 16/28 - Immunoglobulins, e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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
C12N 15/90 - Stable introduction of foreign DNA into chromosome
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12N 15/85 - Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
C40B 30/04 - Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
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.
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.
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.
The present disclosure relates to methods for increasing observed editing rates in the surviving bacteria cells. The compositions and methods presented herein in combination lead to a phenomenon of “edit or die.” Although less cells survive plating and editing, a large percentage of cells that do survive are multiple editors. In one experiment it was found that if a cell survives transformation, plating, and editing, 75% of the surviving cells are multiple editors; that is, 75% of the surviving cells were simultaneously edited with edits at two or more different locations within the bacterial genome.
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.
C12M 3/00 - Tissue, human, animal or plant cell, or virus culture apparatus
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
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
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
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 sphere-packing lattice utilizes lattice-forming beads that are uniform in size and that self-assemble into a crystalline-like lattice.
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 engineered nucleic acid-guided nickases and optimized scaffolds for making rational, direct edits to nucleic acids in live cells.
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.
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
92.
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 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.
C40B 50/06 - Biochemical methods, e.g. using enzymes or whole viable microorganisms
C40B 30/04 - Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
C40B 40/10 - Libraries containing peptides or polypeptides, or derivatives thereof
C07K 16/28 - Immunoglobulins, e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
C07K 14/74 - Major histocompatibility complex [MHC]
C40B 70/00 - Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or barcodes
C12N 15/81 - Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
C12N 15/85 - Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
C12N 15/90 - Stable introduction of foreign DNA into chromosome
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
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
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
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.
The present disclosure provides automated modules and instruments for improved detection of nuclease genome editing of live cells. The disclosure provides improved modules—including high throughput modules—for screening cells that have been subjected to editing and identifying and selecting cells that have been properly edited.
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.
This invention relates to compositions of matter, methods, modules and instruments for automated mammalian cell growth, reagent bundle creation and mammalian cell transfection followed by nucleic acid-guided nuclease editing in live mammalian cells.
