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WO2025022355A1 - Procédés de codage à barres de cellules uniques et de sous-populations cellulaires et leurs utilisations - Google Patents

Procédés de codage à barres de cellules uniques et de sous-populations cellulaires et leurs utilisations Download PDF

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Publication number
WO2025022355A1
WO2025022355A1 PCT/IB2024/057230 IB2024057230W WO2025022355A1 WO 2025022355 A1 WO2025022355 A1 WO 2025022355A1 IB 2024057230 W IB2024057230 W IB 2024057230W WO 2025022355 A1 WO2025022355 A1 WO 2025022355A1
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Prior art keywords
cells
cell
nucleic acid
barcode
population
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Samuel Woodhouse
Liisi LAANISTE
Joachim LUGINBÜHL
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Cosyne Therapeutics Ltd
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Cosyne Therapeutics Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • barcodes are provided herein.
  • molecular tagging compositions for detecting a genetic perturbation, detecting a genetic alteration, detecting an agent, and/or identifying a unique cell lineage within a mixed population of cells.
  • identifying the presence or absence of a genetic alteration in subpopulations of cells in a mixed population of cells wherein: cells of each subpopulation in the mixed population of cells comprise a barcode; and the barcode in each subpopulation of cells is distinct from a barcode in a different subpopulation of cells; the method comprising: (a) isolating nucleic acids from a single cell of the mixed population of cells to obtain isolated nucleic acids, optionally, isolating RNA from the single cell and reverse transcribing the RNA to cDNA; (b) identifying the presence or the absence of the barcode in the cell, wherein the identifying comprises sequencing DNA and/or cDNA, thereby obtaining determined data; (c) processing the determined data from each single cell of the mixed population of cells into groups, wherein each group is assigned to a group according to the barcode, thereby forming aggregated data; and (d) processing the aggregated data for the presence or absence of a genetic alteration in each
  • molecular tagging compositions comprising: (a) a nucleic acid construct, wherein the nucleic acid construct comprises a first barcode sequence; and (b) a set of primers, wherein the set of primers comprise: (i) a 5’ primer nucleic acid, wherein the 5’ primer nucleic acid comprises a sequence that hybridizes to a 5’ sequence of the nucleic acid construct; and (ii) a 3’ primer nucleic acid, wherein the 3’ primer nucleic acid comprises a sequence that hybridizes to a 3’ sequence of the nucleic acid construct.
  • kits for labelling a cell comprising: contacting a cell with the molecular tagging composition provided herein, a nucleic acid construct provided herein, or a vector provided herein, thereby labeling the cell.
  • RNA and RNA are examples of DNA and reverse transcribing the isolated RNA to obtain cDNA.
  • amplifying the isolated DNA and/or the cDNA is also a genetic alteration on a cell-by-cell basis; (e) assigning each cell from the in vitro clonal cell population to a group according to the presence of the same bar
  • FIGS. 1A-1F show schematics of exemplary barcodes and molecular tagging compositions.
  • FIG. 1A shows a schematic of a set of primers binding to genomic DNA comprising a barcode sequence and an agent.
  • FIG. IB shows a schematic of a set of primers that bind an RNA poly-A tail and convert the transcript into cDNA.
  • Primers can be associated with additional identifiers such as a cell barcode (CB) used for assigning transcript to single cells (i.e. for single cell sequencing) and a unique molecular identifier (UMI) used to distinguish individual molecules after sequencing.
  • FIG. 1C shows a schematic of the poly-A and poly-C tail appended to the transcribed RNA. Addition of a barcode and an agent is shown. The construct also contains a capture sequence (CS), upstream of the barcode and perturbation.
  • FIG. ID shows a schematic of a molecular tagging composition that comprises an artificial poly-A sequence inserted downstream of the barcode and perturbation.
  • FIG. IE shows a schematic of several barcodes that permit the association of transcripts to single cells during split-pool barcoding.
  • FIG. IF shows an embodiment of the barcode structure with a combined perturbation sequence (guide RNA).
  • FIGS. 2A-2B shows a schematic of clonal barcoding methods.
  • FIG. 2A shows a typical single-cell RNA-sequencing approach. A mutation is found in some cells, and when clustered by RNA expression, two cell populations are seen.
  • FIG. 2B shows cells marked with a barcode (CB), after single-cell sequencing, can be grouped based on the original starting cell. The pseudo-bulked data can then be used to aggregate all cells within a particular population with the same alteration. Clustering based on RNA expression shows two clusters, but with the additional resolution of the barcode, one of the clusters is identified to contain two cell populations, which are grouped based on genetic alteration.
  • CB barcode
  • FIGS. 3A-3B show schematics of clonal barcoding with genetic perturbation.
  • FIG. 3A shows a typical perturbation approach with single-cell RNA-sequencing read-out. Gene expression changes are determined for each perturbation.
  • FIG. 3B shows cells marked with a barcode called a unique molecular identifier (UMI) that links the barcode to a particular clonal lineage of cells. The cells are also perturbed using an agent. After single-cell sequencing, the cells are grouped based on the original starting cell according to the UMI. The pseudo-bulked data can then be used to label all cells based on the perturbation and the original starting cell. Clustering based on sgRNA shows 3 distinct clusters and 2 clusters grouped based on starting population.
  • UMI unique molecular identifier
  • FIGS. 4A-4C show exemplary schematics of a method to define tumor cell subpopulations by correlating the genetic profiles of pseudo-bulked barcoded cell clones.
  • FIG. 4A shows that clones can be grouped together into one sub-population.
  • FIG. 4B shows the differential gene expression profile between non-targeting and targeting sgRNA perturbations, the response between tumor sub-populations can be studied.
  • Tumor sub-populations can demonstrate highly correlated phenotypes (center panel), indicating a homogenous response to a given gene perturbation.
  • Tumor sub-populations can also exhibit a population-specific response (right panel).
  • FIG. 4C shows that cell viability can also differ on a population and clonal level as a response to a genetic perturbation. Elucidating the genetic alterations associated with the differential response enables discovery of novel synthetic lethal interactions. In this example, clones are significantly reduced in number in response to the genetic perturbation of Gene X2.
  • compositions, methods, and kits for use in the detection of a genetic perturbation, a genetic alteration, an agent, and/or the presence or absence of a barcode (e.g., molecular tag, barcode, or unique molecular tag sequence) in a cell or subpopulations of cells.
  • a barcode e.g., molecular tag, barcode, or unique molecular tag sequence
  • the methods provided herein can be used to resolve genetic diversity and the correlate a perturbation and underlying genetics to aid therapeutic target discovery.
  • a unique cell can be any cell that comprises a barcode of interest, a gene of interest, a genetic alteration, a genetic perturbation, a methylation pattern, or a cellular phenotype.
  • cells that comprise a barcode; a genetic alteration; a genetic perturbation; a barcode and a genetic perturbation; a barcode and a genetic alteration; or any combination thereof can be designated as a unique group of cells and assigned to groups for further characterization (e.g., gene expression, functional analysis, and cellular phenotyping).
  • barcodes and molecular tagging compositions (2) methods of identifying unique cells or cell populations; (3) methods of introducing a genetic perturbation in a nucleic acid; (4) methods of determining the effect of a genetic perturbation; (5) systems; (6) kits; and (7) biotechnology and clinical diagnostic applications.
  • a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range.
  • description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • determining means determining if an element may be present or not (for example, detection). These terms can include quantitative, qualitative or quantitative, and qualitative determinations. Assessing can be alternatively relative or absolute. “Detecting the presence of’ or “identifying the presence of’ are used interchangeably to include determining the amount of something present (e.g., a genetic perturbation, a genetic alteration, or a barcode provided herein), as well as determining whether it may be present or absent.
  • the term “about” a number refer to that number plus or minus: 1%, 2%, 5%, or 10% of that number.
  • the term ‘about’ a range can refer to that range minus: 1%, 2%, 5%, or 10% of its lowest value and plus 10% of its greatest value.
  • affinity molecule or “affinity binding molecule” or “affinity binder” and their grammatical equivalents can be used interchangeably to refer to a molecule, chemical, or an agent that binds to, associates with, or hybridizes to a nucleic acid sequence.
  • the affinity molecule is a primer, a probe, an aptamer, or a solid support.
  • an affinity molecule can be associated with a solid support, for example, through covalent attachment, attachment to a linker associate with the solid support, or non-covalent interactions such as, e.g., hydrogen bonding, ion pairing, hydrophobic or hydrophilic affinity, or Van der Waals interactions.
  • the affinity molecule can bind or hybridize to one or more nucleic acid sequences for identification and detection of the nucleic acids provided herein.
  • the terms “barcode” or “unique molecular tag” or “UMT” or “unique molecular identifier” or “UMI” can be used interchangeably herein to refer to a sequence of nucleotides (for example, DNA or RNA) that are used as an identifier for an associated nucleic acid sequence or as an identifier of the source of an associated molecule, such as a cell-of-origin.
  • a barcode can also refer to any unique nucleic acid sequence, which can be non-naturally occurring- which can, for example, mean not naturally present in a cell, not found in nature, or any combination of these, where the barcode can be used to identify the originating source, of a polynucleotide sequence in a cell, or a cell.
  • a barcode is a random set of nucleotides.
  • the barcodes comprise random nucleotides, semi-random nucleotides, or a combination of random and non-random nucleotides.
  • a barcode is predetermined from a library of barcodes.
  • a library of barcodes include a collection of stored nucleic acid sequences with associated information. Each sequence and the associated information in the library are stored in a database with information such as e.g., the sequence, pattern, cell type, cell subpopulation, promoter type, label type, genetic alteration type, cell group assignment, or epigenetic status.
  • An alteration can be any change in a polynucleotide.
  • a change in a polynucleotide can occur when a molecule associates with, hybridizes to, or intercalates into a polynucleotide or a double stranded polynucleotide.
  • a non-covalent binding e.g., van der Waals interactions, hydrogen bonding, base pairing, wobble base pairing, base stacking
  • an agent such as: an interfering RNA to a polynucleotide, a protein, or a fragment thereof, an antibody or a fragment thereof, an aptamer, an enzyme, or a de-activated enzyme, is an example of an alteration.
  • a protein which can be a CRISPRi protein can bind to the polynucleotide.
  • the introduction of a CRISPR system can result in sequential alterations.
  • the first can be binding of a guide nucleic acid to the polynucleotide.
  • the second can be, when capable of being facilitated by the guide nucleic acid and a biologically active protein (e.g., Cas9), covalent modification of the polynucleotide following binding of the guide to the polynucleotide.
  • a biologically active protein e.g., Cas9
  • an alteration results in a chemical modification of a polynucleotide or a double stranded polynucleotide.
  • Alterations can include chemically modified base of a nucleotide of a polynucleotide (e.g., adding a methyl, hydroxymethyl, formyl, or carboxyl group to the base of the polynucleotide, or hydrolyzing an amino group of an adenosine to form an inosine), a chemically modified sugar of a base of a polynucleotide (e.g., replacing a hydroxyl group with a hydrogen or a methoxy group or a fluorine atom, or forming a locked nucleic acid), or altering a phosphate group or a phosphodiester bond (e.g., to replace phosphorus with sulfur or to replace an oxygen with sulfur).
  • chemically modified base of a nucleotide of a polynucleotide e.g., adding a methyl, hydroxymethyl, formyl, or carboxyl group to the base of the polynucleotide, or hydroly
  • Chemical modification is meant to illustrate changes that can occur on one or more nucleotides of the polynucleotide and is not limited in the way the modification is enacted.
  • a chemical modification such as addition of a methyl epigenetic mark can be affected by an enzyme or a biologically active fragment thereof (e.g., Dmntl).
  • Individual nucleotides of a polynucleotide can be independently chemically modified.
  • chemical modification and covalent modification are used interchangeably.
  • Other exemplary agents that can cause alterations include ethidium bromide, radiation, and enzymes that introduce, alter, or remove epigenetic marks.
  • alterations refers to any intentionally introduced or targeted alteration of a polynucleotide.
  • alterations encompass intentional changes (perturbations) to a polynucleotide, or for example, using a CRISPR-Cas system to add a nucleotide or delete a nucleotide at a pre-determined nucleic acid of a polynucleotide, or binding or hybridizing an interfering RNA to a target RNA represent examples of perturbations because these make targeted changes to a target nucleotide or a fragment thereof.
  • reference sequence can be used to refer to a known nucleotide sequence, e.g., a chromosomal region whose sequence is deposited atNCBFs Genbank database or other databases.
  • a reference sequence can be a wild-type sequence.
  • a reference sequence can be a nucleotide sequence obtained from a healthy individual or a group of healthy individuals without a disease or condition.
  • a “downstream effect” is any resulting change in a cell arising directly or indirectly from an alteration, a perturbation, or both.
  • a targeted deletion of a nucleotide from a polynucleotide can result in a frame-shift resulting in a transcribed RNA that may be converted to an mRNA which codes for a non-functional protein after being translated.
  • the altered coding sequence of the mRNA and the translation of the mRNA to a nonfunctional protein and any loss of function resulting from loss of the functional protein are each effects of the perturbation.
  • a downstream effect can be a phenotypic change in a cell, a tissue, an organ, a system, or an organism.
  • a downstream effect can include altered levels of mRNAs and/or protein.
  • An “agent” provided herein can be any substance (e.g., a chemical, a gene editing system or a component thereof, an alkylating molecule, a nucleic acid, an exogenous polynucleotide that encodes a protein, a plasmid, a vector, a viral vector, a pharmaceutical composition, a protein, an aptamer, a Dmnt, an enzyme, an enzyme inhibitor, a ligand that binds to a receptor or an enzyme, an interfering RNA, a dead Cas enzyme, a CRISPRi enzyme, a mutagen, a repressor, or an enhancer element) or energy (e.g., radiation, X-rays, ultraviolet rays, gamma rays, beta rays, electricity, or heat), an environmental change (e.g., cooling, altering a nutrient or nutrient-supplemented medium), or any combination of these which can be exogenous to a cell,
  • clonal cell can be used to refer to cells that can divide by mitosis into two or more genetically identical daughter cells.
  • a barcode provided herein is passed on from a first cell to any cells that have divided from the first cell (e.g, cell progeny).
  • compositions and barcodes for identifying single cells and identifying unique subpopulations of cells within a mixed population of cells.
  • the barcodes provided herein comprise random nucleotides. In some embodiments, the barcodes comprise semi-random nucleotides. In some embodiments, the barcodes comprise a combination of random and non-random nucleotides. In some embodiments, the barcodes comprise a nucleic acid sequence, that independently comprises from at least about 5 nucleotides to 20 nucleotides in length. In some embodiments, the barcodes comprise a nucleic acid sequence, that independently comprises from at least about 5 nucleotides to 40 nucleotides in length.
  • the barcodes comprise a nucleic acid sequence, that independently comprises from at least about 5 nucleotides to 60 nucleotides in length. In some embodiments, the barcodes comprise a nucleic acid sequence, that independently comprises from at least about 5 nucleotides to 80 nucleotides in length. In some embodiments, the barcodes comprise a nucleic acid sequence, that independently comprises from at least about 5 nucleotides to 100 nucleotides in length.
  • the barcodes provided herein can be synthesized by using a mix of nucleotides during base addition chemical synthesis to create libraries of random sequences (degenerate sequences). They can comprise several such random bases in tandem, with or without known nucleotide sequences intercalated.
  • the barcodes provided herein can comprise 1-, 2-, 3-, 4-, or 5-letter code sequences (e.g., adenine (A); cytosine (C); guanine (G); thymine (T); and optionally, uracil (U) for thymine when the polynucleotide is RNA or when the barcode is a DNA/RNA hybrid). Barcodes can also comprise modifications, unnatural, or degenerate bases.
  • the barcodes and molecular tagging compositions provided herein can be linear or include secondary structure, such as hairpins or loops.
  • a barcode provided herein comprises a nucleic acid sequence comprising deoxyribonucleic acid (DNA).
  • the barcodes comprise ribonucleic acid (RNA).
  • the barcodes comprise a DNA-RNA hybrid.
  • the nucleic acids provided herein comprise an epigenetic mark.
  • the epigenetic mark comprises a methyl group, a hydroxy methyl group, a formyl group, or a carboxyl group.
  • a genetic perturbation or a genetic alteration provided herein is an epigenetic mark.
  • an agent, a perturbation, and/or epigenetic mark can be used to identify a subpopulation of cells within a mixed population of cells.
  • the barcodes are predetermined from a library of barcode sequences associated with information or are random barcodes. In some embodiments, the barcodes are random nucleotide sequences.
  • the barcodes are then added to a nucleic acid sequence in a cell via a vector, electroporation techniques, or chemical transfection (e.g., lipofectamine) .
  • the barcodes are delivered using a cell-specific promoter sequence.
  • Non-limiting examples of promoters include cytomegalovirus (CMV), Rous sarcoma virus (RSV), simian virus 40 (SV40) and mammalian elongation factor la (EFla), cytokeratin 18 and cytokeratin 19, kallikrein, amylase 1C, aquaporin-5 (AQP5), cardiac Troponin-T (cTNT) promoter, Ca2+/calmodulin- dependent kinase subunit a (CaMKII) promoter, neuron-specific enolase (NSE) promoter, synapsin I promoter, synapsin I with a minimal CMV sequence (Synl-minCMV) promoter, glial fibrillary acidic protein (GFAP) promoter, Hb9.
  • CMV cytomegalovirus
  • RSV40 Rous sarcoma virus
  • EFla mammalian elongation factor la
  • the barcodes are configured for single-cell RNA sequencing.
  • a barcode can be introduced via a vector (e.g., a lentivirus) into a cell.
  • a vector e.g., a lentivirus
  • Cells are infected at a low multiplicity of infection (MOI) so that only one lentivirus infects a cell.
  • MOI multiplicity of infection
  • Cells are grown to allow for clonal cell outgrowth, and single-cell analysis is performed. This captures the barcode for a particular clonal lineage and the genetic alterations at the same time. For analysis, cells can then be grouped together if they have the same barcode as this indicates the original starting cell.
  • a barcode can also be introduced to a cell in conjunction with a perturbation in a target polynucleotide.
  • the barcode and an agent that generates a perturbation are introduced to a cell or a population of cells simultaneously.
  • the barcode and an agent that generates a perturbation are introduced to a cell or a population of cells sequentially.
  • the barcode and a guide nucleic acid e.g., a CRISPR guide RNA
  • the barcode and a guide nucleic acid e.g., a CRISPR guide RNA
  • a complex forms between the agent (e.g., the guide nucleic acid), and a target polynucleotide to form a product nucleic acid.
  • the barcode is proximal to an agent in the product nucleic acid.
  • the barcode and the agent are introduced into different polynucleotides of a cell or population of cells.
  • the molecular tagging compositions comprise: a nucleic acid construct comprising a barcode sequence (also referred to herein as a unique molecular tag or a UMT sequence).
  • the nucleic acid construct comprises DNA.
  • the nucleic acid construct further comprises a cell-type specific promoter.
  • the nucleic acid construct further comprises an agent that generates a perturbation in a target nucleic acid. The perturbation can be induced by any method described above or known in the art.
  • the barcode can be a transcribed into RNA when genomic integration occurs, such that the barcode is captured alongside RNA in single-cell sequencing (e.g., in clonal cell populations).
  • the barcode is expressed from the same promoter as the agent in a nucleic acid construct alongside so that within a single transcript there is an agent identifying the gene/target sequence to be altered, a barcode, and optionally, a poly A sequence for RNA sequencing.
  • the poly A sequence can also be replaced by a defined capture sequence.
  • molecular tagging compositions comprising a set of primers.
  • the set primers comprise: a 5’ primer nucleic acid.
  • the 5’ primer nucleic acid comprises a sequence that hybridizes to a 5’ sequence of the nucleic acid construct.
  • the set of primers comprise: a 3’ primer nucleic acid.
  • the 3’ primer nucleic acid comprises a sequence that hybridizes to a 3’ sequence of the nucleic acid construct.
  • the set of primers comprise DNA, RNA, or a combination thereof.
  • the nucleic acid construct or the set of primers further comprises a chemical modification, a colorimetric tag, a photoreactive tag, a phase changing agent, an aptamer, a protein, an antibody, or an primer nucleic acid sequence.
  • the set of primers comprise a chromophore.
  • a nucleic acid provided herein can be modified with a linker or spacer, e.g.., at an internal position, on the 3’- and/or 5 ’-end.
  • the linker or spacer is used for linking the nucleic acid strand with a moiety, such as a solid support or a tag provided herein.
  • the linker or spacer can be selected from the group consisting of photocleavable linkers, hydrolysable linkers, redox cleavable linkers, phosphate -based cleavable linkers, acid cleavable linkers, ester- based cleavable linkers, peptide-based cleavable linkers, and any combinations thereof.
  • the cleavable linker can comprise a disulfide bond, a tetrazine-trans-cyclooctene group, a sulfhydryl group, a nitrobenzyl group, a bromo hydroxycoumarin group, a bromo hydroxy quinoline group, a hydroxyphenacyl group, a dimethoxybenzene group, or any combinations thereof.
  • the cleavable linker can comprise a photocleavable linker.
  • the 5’ primer nucleic acid and/or the 3’ primer nucleic acid further comprise a sequencing probe or an affinity molecule.
  • the 5’ primer nucleic acid or the 3’ primer nucleic acid comprise an affinity molecule for nucleic acid isolation and capture.
  • a nucleic acid provided herein is contacted with a solid support (e.g., streptavidin), thereby isolating the nucleic acid.
  • the nucleic acid construct or the set of primers comprise an element that permits detection of the nucleic acid by a detector. Any modifications to the nucleic acid constructs, primers, or products provided herein that permit purification, extraction, quantification of expression, binding, electrophoresis, and the like, can also be made.
  • the nucleic acid construct further comprises a one or more additional barcode.
  • a molecular tagging composition provided herein further comprises an additional nucleic acid construct provided herein, and wherein the additional nucleic acid construct, optionally comprises an additional barcode sequence. Multiple barcodes can be used in the same nucleic acid construct or in different nucleic acid constructs depending on the cellular identification method being used.
  • a product nucleic acid when the set of primers hybridize to the nucleic acid construct, a product nucleic acid is formed.
  • the product nucleic acid binds to one or more sequencing probe proximal to the first barcode (or unique molecular tag (UMT)) sequence for cell population analysis.
  • UMT unique molecular tag
  • the molecular tagging composition further comprises a second nucleic acid construct comprising an agent that generates a perturbation in a target nucleic acid.
  • the molecular tagging composition further comprises a second barcode (or unique molecular tag (UMT)).
  • the molecular tagging composition further comprises a second set of primers. In some embodiments, the second set of primers hybridize to the second nucleic acid construct.
  • the second set of primers hybridize to the second nucleic acid construct to form a second product nucleic acid.
  • the second product nucleic acid binds to a sequencing probe assigned to the second barcode sequence for cell population analysis.
  • RNA polymerases typically include eukaryotic RNA polymerases I, II, and III, and bacterial RNA polymerases as well as phage and viral polymerases. RNA polymerases can be DNA-dependent and RNA-dependent.
  • vectors comprising the molecular tagging compositions provided herein or a nucleic acid construct provided herein.
  • the nucleic acid construct is at least a portion of a sequence in a plasmid.
  • the vector is a minicircle vector.
  • the vector is a viral vector.
  • the viral vector comprises an adeno-associated virus (AAV), a recombinant AAV, a lentivirus, an alphavirus.
  • AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, e.g., the rep and/or cap genes, but retain functional flanking ITR sequences.
  • the host cell is rendered capable of encoding AAV polypeptides that are required for packaging the AAV vector (containing a recombinant nucleotide sequence of interest) into infectious recombinant virion particles for subsequent gene delivery.
  • the AAV or the rAAV provided herein comprises a serotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrhlO, or any combination thereof.
  • cells comprising a molecular tagging composition provided herein, or a nucleic acid construct provided herein. Methods of identifying the barcodes/UMT sequences, genetic alterations, agents, and genetic perturbations in a cell or cell population are further described in detail below.
  • the cells of each subpopulation in the mixed population of cells comprise a barcode.
  • the barcode is introduced into a cell population via a vector.
  • the barcode in each subpopulation of cells is distinct from a barcode in a different subpopulation of cells.
  • the mixed population of cells or a subpopulation of cells are contacted with the barcode, a plurality of barcodes, or a molecular tagging composition provided herein.
  • the mixed population of cells are cultured for a period of time in culture.
  • the methods comprise culturing the mixed population of cells in a first cell culture medium for a period of time and contacting the mixed population of cells with the barcode.
  • the methods comprise culturing a plurality of subpopulations of cells separately prior to admixing the subpopulations into a single container, vessel, or arrangement (e.g., for cell and tissue engineering applications).
  • the methods comprise culturing a plurality of subpopulations of cells in a first cell culture medium for a period of time and contacting each subpopulation of cells separately with the barcode.
  • each subpopulation of cells comprises a different barcode.
  • the subpopulations of cells are admixed in a second cell culture medium for a period of time.
  • the cells can be cultured for any period of time that permits clonal outgrowth of the initial subpopulation of cells contacted with a gene editing system provided herein and/or a barcode provided herein.
  • the cells are cultured for a period of time in a cell culture medium, wherein the period of time is at least about 5 hours, at least about 10 hours, at least about 12 hours, at least about 15 hours, at least about 20 hours, at least about 24 hours (1 day), at least about 48 hours (2 days), at least about 72 hours (3 days), at least about 96 hours (4 days), at least about 120 hours (5 days), at least about 144 hours (6 days), at least about 168 hours (7 days), at least about 336 hours (14 days), at least about 504 hours (21 days), at least about 672 hours (28 days), up to 744 hours (31 days).
  • the cells are cultured longer than 744 hours (e.g., 1 month).
  • the mixed population of cells or the subpopulations of cells comprise prokaryotic cells, eukaryotic cells, or both.
  • a mixed population of cells can include a plurality of two or more different cell types, cell subtypes, or cell lineages.
  • the mixed population of cells comprise: a tissue, a clonal cell population, a population of cells isolated from a subject, a population of genetically modified cells, a population of dissociated cells, a population of cells that have been contacted with an agent, a population of in vitro- differentiated cells, a population of stem cells, a population of fibroblasts, a population of blood cells, a population of immune cells, a population of tumor cells isolated from a subject, or any combination thereof.
  • the mixed population of cells or the subpopulations of cells comprise mammalian cells.
  • the mixed population of cells or the subpopulations of cells comprise human cells.
  • the cells provided herein are contacted with an agent prior to isolation of nucleic acids. In some embodiments, the cells provided herein are contacted with an agent after cells are cultured for a period of time.
  • the agent is a chemical, a molecule, a salt, an analyte, a protein, an antibody or an antibody fragment, a nucleic acid, a guide nucleic acid, a plasmid, a vector, a viral vector, or a gene editing system.
  • the agent is a gene editing system provided herein or components associated with a gene editing system (e.g., a guide nucleic acid and a Cas protein).
  • the methods comprise: isolating nucleic acids from a single cell, a subpopulation of cells, or a mixed population of cells to obtain isolated nucleic acids.
  • the methods comprise: crosslinking a cell, a subpopulation of cells, or a mixed population of cells.
  • the crosslinked cell, the crosslinked subpopulation of cells, or the crosslinked mixed population of cells are contacted with a reverse transcriptase enzyme to produce cDNA.
  • a single cell, a subpopulation of cells, or a mixed population of cells are live cells (e.g., that are not fixed with a crosslinking agent).
  • the isolated nucleic acids comprise DNA, RNA, or a combination of DNA and RNA.
  • the isolated nucleic acids comprise RNA, wherein the RNA is a coding RNA, a mRNA, a pre-RNA, a miRNA, an siRNA, a tRNA, a piRNA, a rRNA, a tmRNA, a double stranded RNA, a ssRNA, an snRNA, a snoRNA, IncRNA, or any combination thereof.
  • the methods comprise isolating RNA from the single cell and reverse transcribing the RNA to generate cDNA.
  • the cDNA or the isolated DNA are purified.
  • the isolated nucleic acids are fragmented.
  • the isolated nucleic acids or the cDNA are amplified by polymerase chain reaction (PCR).
  • the methods provided herein further comprise identifying the presence or the absence of the barcode in the cell.
  • the identifying comprises contacting the isolated DNA or the isolated cDNA with an affinity molecule and sequencing DNA, thereby obtaining determined data. Sequences reveal combined barcode sequence and target sequence information.
  • the sequencing comprises DNA sequencing, next generation sequencing, high-throughput sequencing, nanopore sequencing, long read sequencing, Sanger sequencing, Maxam-Gilbert sequencing, methylation sequencing, or any combination thereof.
  • the sequencing readouts can then be used to define cell types, cell lineage, assign barcode groups, and identify a genetic alteration or an agent (e.g., a guide RNA sequence), in a target nucleic acid sequence.
  • the methods provided herein comprise processing steps for identifying the presence or absence of a barcode, a genetic perturbation, an agent (e.g., an agent that generates a perturbation in a cell) and/or a genetic alteration in a cell or subpopulation of cells to obtain determined data.
  • the determined data from each single cell of the mixed population of cells is processed into groups.
  • each group is assigned to a group according to the barcode, thereby forming aggregated data.
  • the aggregated data is processed for the presence or absence of a genetic alteration in each group from step (c), thereby identifying the presence or absence of a genetic alteration in a subpopulation of cells in a mixed population of cells.
  • the processing can be performed by a computer or a device comprising a processor and a computer readable memory.
  • the methods comprise contacting a cell, a population of cells, or a mixed population of cells with an agent.
  • the agent can be an agent that indirectly or directly generates a genetic perturbation or a genetic alteration in a nucleic acid of a cell or cellular genome.
  • the agent comprises a chemical, a protein, an antibody or an antibody fragment, a nucleic acid, a plasmid, a vector, a viral vector, a transcriptional regulator, a nucleic acid encoding a transcriptional regulator, a gene editing system, a nucleic acid encoding a gene editing system, a nucleic acid encoding a guide nucleic acid (e.g., a guide RNA), or an interfering RNA (RNAi).
  • the RNAi comprises an silencing RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA).
  • the agents provided herein can be formulated for delivery to a cell, population of cells, or a tissue.
  • the agent is in complex with a carrier (e.g., a nanoparticle, a liposome, an extracellular vesicle, or a lipid carrier).
  • the agent is encapsulated by a carrier.
  • the agent comprises a vector comprising a nucleic acid, a guide nucleic acid, a gene editing system, or any combination thereof.
  • the methods comprise contacting a cell, a population of cells, or a mixed population of cells with a gene editing system or a nucleic acid encoding a gene editing system.
  • the gene editing system comprises: a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system, a catalytically inactive endonuclease, a Transcription Activator-Like Effector Nucleases (TALENS), a transposon systems (e.g., Sleeping Beauty), a zinc finger nuclease (ZFN), a meganuclease, a fusion protein, a derivative, a variant, or a mutant thereof.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • TALENS Transcription Activator-Like Effector Nucleases
  • ZFN zinc finger nuclease
  • a database can identify regions of a target nucleic acid sequence that are permissive to genetic engineering depending on the method of editing.
  • a database can be ENCODE (encyclopedia of DNA Elements) (available on the world wide web at genome. gov/10005107), CHOP tool (available on the world wide web at chopchop.cbu.uib.no), GenomeCRISPR (available on the world wide web at genomecrispr.org), the Basic Local Alignment Search Tool (BLAST) (available on the world wide web at blast.ncbi.nlm.nih.gov/Blast.cgi), or the Sanger Institute Genome Editing (WGE) (available on the world wide web at wge.stemcell.sanger.ac.uk).
  • ENCODE encyclopedia of DNA Elements
  • CHOP tool available on the world wide web at chopchop.cbu.uib.no
  • GenomeCRISPR available on the world wide web at genomecrispr.org
  • Targeted genetic perturbations can begin from the generation of nuclease-induced double-stranded breaks (DSBs) or single stranded breaks, that lead to the stimulation of highly efficient recombination mechanisms of cellular DNA in mammalian cells.
  • Double strand breaks are introduced into a cell using one or more endonucleases (e.g., S. pyogenes Cas9 deoxyribonucleic acid (DNA) endonuclease) and one or two nucleic acid guide sequences to affect a pair of double-strand breaks (DSBs).
  • the gene editing system comprises one or more guide nucleic acid.
  • the one or more guide nucleic acid comprises an RNA, a DNA, or a combination thereof.
  • the one or more guide nucleic acid is a DNA-RNA hybrid.
  • the gene editing system is a CRISPR system.
  • a CRISPR system can comprise an endonuclease and one or more guide RNAs.
  • the endonuclease is selected from the group consisting of: Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Casl l, Casl2, Casl3, Casl4, Csyl , Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl,
  • an endonuclease induces a genetic perturbation in a target nucleic acid sequence (e.g., an oncogene or a proto-oncogene).
  • targeting of the genetic perturbation is determined by identifying where a protospacer adjacent motif (PAM) sequence is located in a target sequence.
  • the PAM sequence is capable to hybridizing to a portion of a guide nucleic acid.
  • a genetic perturbation can be within about 20 base pairs, about 10 base pair, about 5 base pairs, about 3 base pairs, or about 2 base pairs from a protospacer adjacent motif (PAM) sequence.
  • a PAM can be a nucleotide sequence within gene, genome, or chromosome that is targeted by a guide RNA.
  • the site of cleavage by an RNA-guided nuclease is within a protospacer sequence or adjacent to the sequence.
  • the Cas protein will generate a double strand break within the protospacer sequence, thereby cleaving the protospacer and disrupting the target gene sequence.
  • disruption of the protospacer can result though non-homologous end joining (NHEJ) or homology-directed repair (HDR).
  • NHEJ non-homologous end joining
  • HDR homology-directed repair
  • Disruption of the protospacer can result in the deletion of the protospacer and/or target gene.
  • disruption of the protospacer can result in an exogenous nucleic acid sequence being inserted into the protospacer or replacing the protospacer sequence.
  • the methods comprise contacting the mixed population of cells with a CRISPR interference (CRISPRi) system or a CRISPR activation (CRISPRa) system.
  • CRISPRi CRISPR interference
  • CRISPRa CRISPR activation
  • the CRISPRi system comprises a catalytically dead or deactivated endonuclease enzyme e.g., a dead Cas9 or dCas9) fused to one or more repressor proteins (e.g., SALL1 or SDS3).
  • the CRISPRi system further comprises a guide RNA, wherein the guide RNAs binds to a target sequence of a polynucleotide that is upstream or downstream of a transcriptional start site (e.g., within 50 base pairs).
  • the guide in a CRISPRi system can associate the enzyme-repressor protein fusion protein and direct the repressor domain of the protein to the target sequence in a polynucleotide, thereby inhibiting transcription of the polynucleotide.
  • the CRISPRa system comprises a catalytically dead or deactivated endonuclease enzyme (e.g., dCas9) fused to a transcriptional activator (e.g., Vp64).
  • the CRISPRa system comprises a guide RNA.
  • the guide RNA of the CRISPRa system binds to a target sequence in a polynucleotide, wherein the target sequence is upstream of a promoter or the transcriptional start site.
  • the guide in a CRISPRa system can associate the enzyme-activator fusion protein domain of the fusion protein to the target activation sequence in a polynucleotide, thereby activating transcription of the polynucleotide.
  • the presence of a guide RNA sequence in a cell indicates that the cell comprises a genetic perturbation that was introduced by the guide RNA.
  • the genetic perturbation is induced by site-directed mutagenesis.
  • Site-directed mutagenesis is a method of creating specific mutations in a known nucleic acid sequence. The method can be performed using traditional polymerase chain reaction (PCR) techniques, primer extension mutagenesis, or inverse PCR mutagenesis.
  • PCR polymerase chain reaction
  • compositions and methods that can introduce a genetic perturbation include: contacting a cell or a population of cells with an analyte, a chemical, an antisense oligonucleotide, a ribozyme, an enzyme, a transcriptional repressor, an enhancer, a gene editing system, a CRISPRi system, a CRISPRa system, a morpholino, a transgene, or a transposon system; or exposing a cell or a population of cells to various wavelengths of light, UV light, X-rays, gamma rays, mechanical stimulation, or electrical stimulation.
  • the methods provided herein comprise: (a) isolating nucleic acids from a single cell of the mixed population of cells to obtain isolated nucleic acids, optionally, isolating RNA from the single cell and reverse transcribing the RNA to cDNA; (b) identifying the presence or the absence of the barcode in the cell, wherein the identifying comprises sequencing DNA and/or cDNA, thereby obtaining determined data; (c) processing the determined data from each single cell of the mixed population of cells into groups, wherein each group is assigned to a group according to the barcode, thereby forming aggregated data; and (d) processing the aggregated data for the presence or absence of a genetic alteration in each group from step (c), thereby identifying the presence or absence of a genetic alteration in a subpopulation of cells in a mixed population of cells.
  • steps (a)-(d) are performed consecutively.
  • the methods further comprise: (i) a step of quantifying a level of expression of a gene relative to a reference in the mixed population of cells; (ii) assigning each cell to a group according to the level of expression of a gene relative to a reference in the mixed population of cells; or (iii) both (i) and (ii).
  • the methods comprise (a) contacting a mixed population of cells with an agent that modulates a target nucleic acid in a cell within the mixed population of cells, thereby introducing a genetic perturbation in a target nucleic acid sequence; (b) contacting the mixed population of cells with a plurality of barcode sequences or a plurality of molecular tagging compositions that comprise a barcode sequence; (c) culturing the mixed population of cells for a period of time that permits a cellular division; (d) isolating DNA from the mixed population of cells or isolating RNA from the mixed population of cells and reverse transcribing the isolated RNA to cDNA; (e) sequencing the DNA and/or cDNA, thereby obtaining determined data; (f) processing the determined data from each single cell of the mixed population of cells into groups, wherein each group is assigned to a group according to the barcode
  • the methods provided herein can be used to determine the gene editing efficiency of a gene editing system.
  • Provided herein are methods of quantifying gene editing efficiency of a gene editing system, where the methods comprise contacting a mixed population of cells with a gene editing system or a nucleic acid encoding a gene editing system to introduce a genetic perturbation in the genome of a cell or a subpopulation of cells within a mixed population of cells; and contacting the mixed population of cells with one or more barcodes.
  • the methods provided herein comprise culturing a population of cells for a period of time.
  • the period of time is at least about 24 hours (1 day), at least about 48 hours (2 days), at least about 72 hours (3 days), at least about 96 hours (4 days), at least about 120 hours (5 days), at least about 144 hours (6 days), at least about 168 hours (7 days), at least about 336 hours (14 days), at least about 504 hours (21 days), at least about 672 hours (28 days), up to 744 hours (31 days) or a period of time that permits a cell division of a subpopulation of cells in the mixed population of cells.
  • the methods further comprise counting the number of viable cells in each subpopulation.
  • the methods comprise isolating nucleic acids from the mixed population of cells; isolating DNA from the mixed population of cells or isolating RNA from the mixed population of cells and reverse transcribing the isolated RNA to cDNA.
  • the methods comprise a step of amplifying the isolated DNA or the isolated cDNA to form amplicons. Methods of forming amplicons, include, e.g., polymerase chain reaction techniques.
  • the methods comprise identifying the presence or the absence of a barcode in the cell.
  • the isolating step and the identifying steps are conducted on a cell-by-cell basis.
  • the barcode can be identified by sequencing to obtain determined data, sequencing the plurality of tagged nucleic acid molecules or derivatives thereof to generate sequence reads, wherein the sequence reads comprise the sequence of the nucleic acid molecule and the sequence of the associated barcodes.
  • the methods comprise aligning the sequence reads to a reference sequence wherein the starting position and ending position of the sequence of the nucleic acid molecule from which the reads are derived can be determined.
  • the aligning is performed by a computer implemented method, wherein the reference sequence provides a sense strand and an anti-sense strand information.
  • the methods comprise determining which reference sequence the sequence reads align to in a library of reference sequences.
  • the methods comprise identifying reads with the same barcode that can be assigned to the same cell lineage via the genetic alteration, the agent, or the genetic perturbation introduced by the agent.
  • the methods comprise determining a count number of the nucleic acids that have been genetically altered and/or comprise a barcode.
  • the sequence can be aligned to the same reference sequence location by a computer implemented method.
  • the methods comprise processing the determined data from each single cell of the mixed population of cells into groups, wherein each group is assigned to a group according to a barcode, wherein the barcode in each group is distinct from a barcode in a different group, thereby forming aggregated data.
  • the methods comprise processing the aggregated data for the presence or absence of a genetic perturbation in each group.
  • the methods comprise processing the aggregated data for the presence or absence of an agent in each group.
  • the agent is a guide RNA or a fragment thereof.
  • the methods comprise quantifying the number of cells that comprise a barcode
  • the methods comprise processing the aggregated data for the presence or absence of a genetic alteration in each group.
  • the methods comprise quantifying the number of cells that comprise an agent and/or a genetic perturbation.
  • the methods comprise quantifying the number of cells that comprise both a genetic alteration and a barcode.
  • the methods comprise quantifying the number of cells that comprise both a genetic perturbation and a barcode.
  • the methods comprise quantifying the number of cells that comprise agent and a barcode.
  • the methods comprise quantifying the number of cells that comprise a genetic perturbation, a genetic alteration, an agent, a barcode, a downstream effect, or any combination thereof. In some embodiments, the methods comprise quantifying the percentage of cells that comprise the barcode and the agent or the genetic alteration relative to the population of cells that have the same barcode and do not comprise the agent or the genetic alteration, thereby quantifying the gene editing efficiency of the gene editing system.
  • the gene editing system comprises a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system.
  • CRISPR/Cas system comprises one or more guide nucleic acid and an endonuclease selected from the group consisting of: Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Cas 11, Cas 12, Casl3, Casl4, Cbfl, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpfl (or Casl2a), Csbl, Csb2, Csb3, Csxl7, Csxl4,
  • the endonuclease is catalytically inactivated (also referred to herein as a dead or deactivated endonuclease (e.g., a dead Cas enzyme).
  • the gene editing system introduces a genetic perturbation in a cell.
  • the gene editing system further introduces a genetic alteration in a cell.
  • the genetic alteration is an off-target effect of the gene editing system.
  • the genetic alteration is an indirect effect of the gene editing.
  • the genetic perturbation comprises an insertion, a deletion, a change in copy number, a point mutation, a missense mutation, a nonsense mutation, a mutation in a stop codon, an epigenetic mark, a reduction in gene expression, an overexpression of a gene, or any combination thereof.
  • the genetic perturbation or the genetic alteration is comprised in an oncogene.
  • the agent that introduces the genetic perturbation is comprised in a promoter.
  • the methods comprise a step of identifying the subpopulation or subpopulations of cells that comprise a genetic alteration induced by the gene editing system and an agent. In some embodiments, the methods comprise counting the number of viable cells in each subpopulation after the cells have been contacted with an agent or a gene editing system.
  • the methods comprise a step of quantifying a level of expression of a gene relative to an appropriate control.
  • a level of expression of a gene is determined by determining a number of DNAs, RNAs, cDNAs, or any combination thereof.
  • the processing steps are performed by computer or a device comprising a processor and a computer readable memory.
  • detecting, determining, identifying steps provided herein comprise a computer-implemented method. For example, determining the presence or the absence of a genetic alteration, a genetic perturbation, or a barcode provided herein can be determined by a computer implemented method. In some instances, a computer- implemented method can be executed on a computing device.
  • a computing device can comprise a memory, one or more executable instructions, and/or one or more processors operably connected to the memory, wherein the one or more processors are configured to execute the one or more executable instructions.
  • a computing device can comprise one or more computer modules.
  • a computing device can comprise a mobile device, a stationary device, a personal computer, a desktop computer, a tablet computer, a laptop computer, a smart device, or any combination thereof.
  • a computing device can be connected to the internet, a secure network, a private network or a combination thereof.
  • a computing device can be operably connected to a speaker, a camera, a printer, an input device (e.g., a mouse or a keyboard), a screen, a video display or any combination thereof.
  • a computing device can comprise a graphical user interface (GUI).
  • GUI graphical user interface
  • data obtained from determining the presence or absence of a deletion can be transmitted through a network.
  • a non-transitory computer storage medium storing instructions can be operable when executed by one or more processors to implement a method described herein.
  • a system can comprise a computer-implemented method described herein.
  • a computer-implemented method can be used for determining the presence or absence of a deletion of a region of DNA.
  • a computer-implemented method can be used for determining the level or amount of DNA relative to an appropriate control sample.
  • a storage medium can store computer instructions (e.g., computer codes) which can be operable when executed by one or more processors performing a method for identifying the presence or absence of a genetic perturbation, a genetic alteration, or a barcode provided herein.
  • computer instructions e.g., computer codes
  • devices for use in sequencing, cell sorting, cell counting, cell imaging, and gene expression analysis.
  • Devices provided herein can include, for example, microfluidic devices.
  • the microfluidic device can be used to divert a solution of barcodes along multiple spatially separated directions or to different containers of cell subpopulations. This may be used to analyze each of several possible barcodes and genetic alterations individually.
  • multiple verifications can be carried out on the same mixed population of cells, and the resultant pattern can be validated by sequencing, fluorescent, or color change readouts, e.g., by the naked eye or a device, e.g., a microscope and computer.
  • microfluidic channels can be constructed of glass, biocompatible plastic, rubber, metal, or any combination thereof.
  • the nucleic acids provided herein can be conjugated to a solid support.
  • the solid support can be used to capture or label cells, proteins, nucleic acids, primer sequences, barcodes, or molecular tagging compositions.
  • the solid support can exist in the form of a platform, column, filter or sheet, dish, a microfluidic capture device, capillary tube, electrochemical responsive platform, scaffold, cartridge, resin, matrix, bead, or another solid support known in the art.
  • the solid support comprises materials that include, but are not limited to, a polymer, metal, ceramic, gels, paper, or glass.
  • the materials of the solid support can further comprise, as non-limiting examples, polystyrene, agarose, gelatin, alginate, iron oxide, stainless steel, gold nanobeads or particles, copper, silver chloride, polycarbonate, polydimethylsiloxane, polyethylene, acrylonitrile butadiene styrene, cyclo-olefin polymers or cyclo-olefin copolymers, or SepharoseTM resin.
  • the solid support is magnetoresponsive (e.g., magnetite, iron (III) oxide, samarium-cobalt, terfenol-D, or any other magnetic element).
  • the kit comprises a unit comprising a reagent for carrying out nucleic acid isolation. In some embodiments, the kit comprises reagents for RNA isolation and reverse transcription reactions. In some embodiments, the kit comprises cell culture medium. In some embodiments the kit comprises reagents for sequencing nucleic acids. In some embodiments, the kit comprises a tag, adapter sequences, a set of primers, and/or an affinity probe.
  • a molecular tagging composition described provided herein is prepared in a single container for contacting a plurality of cells.
  • a composition provided herein is prepared in two containers, separating the nucleic acid construct from the barcodes.
  • “container” includes vessel, vial, ampule, tube, cup, box, bottle, flask, jar, dish, well of a single-well or multi -well apparatus, reservoir, tank, or the like, or other device in which the herein disclosed compositions may be placed, stored and/or transported, and accessed to remove the contents. Examples of such containers include glass and/or plastic sealed or re-sealable tubes and ampules. In some embodiments, the containers are RNase free and/or DNase free.
  • the barcodes provided herein and the gene editing system or components thereof are in different vectors. In some embodiments, the barcode and the gene editing system provided herein are in different containers.
  • the methods comprise contacting a cell, a subpopulation of cells, or a mixed population of cells with a molecular tagging composition provided herein, a barcode provided herein, or a nucleic acid construct provided herein, thereby labeling the cell, the subpopulation of cells, or the mixed population of cells with a label.
  • the method of labeling can be used to track single cells within a mixed population of cells, group cells into subpopulations using the unique barcode sequences, and identify the presence or absence of a unique barcode in combination with one or more of: an agent, a genetic perturbation, a genetic alteration, a downstream effect, or a combination thereof.
  • RNA-sequencing RNA-sequencing
  • RNA-sequencing Single-cell RNA-sequencing
  • After contacting a population of cells with an agent or a gene editing system cells are allowed to proliferate for a period of time (e.g., 7 to 10 days).
  • the methods provided herein enables deconvolution of the population of cells and identifies clonal phenotypes for cell subpopulations that comprise a particular barcode and/or genetic alteration.
  • the genetically perturbed cells can be identified for a genetic alteration phenotype that is unique to a specific clone, unique to specific subsets, and/or a phenotype that is common to all cells within a tested mixed cell population.
  • kits for identifying the presence or absence of a genetic alteration in an in vitro clonal cell population comprising: contacting the clonal cell population with one or more molecular tagging composition provided herein; isolating DNA on a cell-by-cell basis from a plurality of cells of the clonal cell population to obtain isolated DNA.
  • the methods comprise isolating RNA on a cell-by-cell basis to form isolated RNA and reverse transcribing the isolated RNA to obtain cDNA.
  • the methods comprise amplifying the isolated DNA or the cDNA to form amplicons.
  • the methods comprise identifying the presence or absence of the genetic alteration in the plurality of clonal cells, and the presence or absence of a barcode sequence, wherein the presence of the genetic alteration and the presence of a barcode sequence in at least about 10% of the plurality of cells indicates the presence of the genetic alteration in the in vitro clonal cell population.
  • the clonal cell population comprises a population of cancer cells.
  • the methods comprise: contacting a cell, a population of cells, or a tissue (e.g., a tumor or a population of cancer cells) with an agent; contacting the cell, population of cells, or tissue with one or more molecular tagging composition provided herein; isolating DNA on a cell-by-cell basis from a plurality of cells of the clonal cell population to obtain isolated DNA.
  • a tissue e.g., a tumor or a population of cancer cells
  • the agent is a gene editing system, a gene silencing system, a gene activating system, or a component thereof.
  • the agent is a chemical, a nucleic acid, an antibody, a protein, or any combination thereof.
  • the method further comprises quantifying cell viability before and after contacting the tumor or the population of cells with the agent.
  • the methods provided herein can be used to pair a transcriptional response (e.g., tumor sternness) or cellular phenotype (e.g., cell death) and disease-associated genetic features with greater resolution and accuracy than previous methods.
  • identifying the presence or absence of a genetic alteration in subpopulations of cells in a mixed population of cells wherein: cells of each subpopulation in the mixed population of cells comprise a barcode; and the barcode in each subpopulation of cells is distinct from a barcode in a different subpopulation of cells; the method comprising: (a) isolating nucleic acids from a single cell of the mixed population of cells to obtain isolated nucleic acids, optionally, isolating RNA from the single cell and reverse transcribing the RNA to cDNA; (b) identifying the presence or the absence of the barcode in the cell, wherein the identifying comprises sequencing DNA and/or cDNA, thereby obtaining determined data; (c) processing the determined data from each single cell of the mixed population of cells into groups, wherein each group is assigned to a group according to the barcode, thereby forming aggregated data; and (d) processing the aggregated data for the presence or absence of a genetic alteration in each
  • molecular tagging compositions comprising: a nucleic acid construct, wherein the nucleic acid construct comprises a first barcode sequence, and optionally, a sequence comprising an agent. Further provided herein are molecular tagging compositions comprising a set of primers.
  • molecular tagging compositions wherein the set of primers comprise: (i) a 5’ primer nucleic acid, wherein the 5’ primer nucleic acid comprises a sequence that hybridizes to a 5’ sequence of the nucleic acid construct; and (ii) a 3’ primer nucleic acid, wherein the 3’ primer nucleic acid comprises a sequence that hybridizes to a 3’ sequence of the nucleic acid construct.
  • molecular tagging compositions comprising: (a) a nucleic acid construct, wherein the nucleic acid construct comprises a first barcode sequence, and optionally, a sequence comprising an agent, wherein the genetic perturbation was introduced to the nucleic acid construct by an agent; and (b) a set of primers, wherein the set of primers comprise: (i) a 5’ primer nucleic acid, wherein the 5’ primer nucleic acid comprises a sequence that hybridizes to a 5’ sequence of the nucleic acid construct; and (ii) a 3’ primer nucleic acid, wherein the 3’ primer nucleic acid comprises a sequence that hybridizes to a 3’ sequence of the nucleic acid construct.
  • vectors comprising the molecular tagging composition provided herein or the nucleic acid construct provided herein.
  • cells wherein the cells comprise a molecular tagging composition provided herein or a nucleic acid construct provided herein.
  • kits for labelling a cell comprising: contacting a cell with the molecular tagging composition provided herein, a nucleic acid construct provided herein, or a vector provided herein, thereby labelling the cell.
  • RNA and reverse transcribing the isolated RNA to obtain cDNA are examples of molecular tagging compositions provided herein;
  • identifying the presence or absence of the genetic alteration, a barcode sequence, an agent, or a genetic perturbation introduce by the agent on a cell-by-cell basis are examples of the genetic alteration, a barcode sequence, an agent, or a genetic perturbation introduce by the agent on a cell-by-cell basis;
  • Example 1 Barcode Structure, Function, and Methods of Use.
  • FIGS. 1A-1F illustrate several barcodes, product nucleic acids, and methods of integrating barcodes into a target nucleic acid.
  • a barcode can be inserted into a nucleic acid construct under a promoter that ensures that the barcode is transcribed into RNA.
  • a barcode can be a pre-determined sequence from a library of random barcodes or contain random or semi-random nucleotide sequences.
  • a viral vector e.g., a lentivirus or AAV
  • a viral vector can be used to insert the barcode sequence into the genome of a cell or population of cells via an integration method.
  • a plasmid is used to integrate the barcode sequence via recombination into the cell (e.g., using piggy -bac).
  • a selfreplicating RNA can be used to introduce the barcode into a cell.
  • a genetic perturbation can be introduced into a cellular genome.
  • the agent that introduces the genetic perturbation e.g., a guide RNA and CRISPR system; or an RNAi
  • one or more barcode sequences can be controlled under the same promoter or different promoters.
  • the barcode and the agent are on delivered to a population of cells via different nucleic acid strands, different plasmids, and/or different vectors.
  • the barcode can also be inserted without an agent as a label for clonal cells.
  • the barcode sequence can be captured using single-cell sequencing approaches.
  • RNA can be isolated, and reverse transcribed to cDNA for detection or sequencing. In this case, the barcode sequence is retained when cells undergo cell division to permit clonal tracking.
  • a barcode and the genetic perturbation are captured without the need for transcription into RNA using primers binding to genomic DNA upstream and downstream of the barcode and genetic perturbation (FIG. 1A).
  • the genetic perturbation can be introduced by an agent such as a CRISPR system, an RNAi, or any agent that introduces a change in the genome that differs from a reference DNA sequence.
  • a reference sequence can be found in a database (e.g., the NIH National Center for Biotechnology Information, also referred to as NCBI) or a control sequence prior to the introduction of the genetic perturbation identified by next generation sequencing.
  • a set of primers are introduced and associate with the 5’ and 3’ ends of the target nucleic acid for next generation sequencing.
  • the resulting amplicon contains 5’ and 3’ sequencing primers, the genetic perturbation that was introduced by the agent, and the barcode sequence (FIG.
  • the perturbed DNA construct is transcribed into RNA, during which the cell’s own transcriptional machinery appends a 5’ poly-G cap and a 3’ poly- A tail (FIG.
  • primers can be associated with additional identifiers such additional cell barcodes (denoted in FIG. IB as “CB”) used for assigning the transcript to single cells (i.e.
  • a unique molecular identifier sequence (denoted as “UMI”) is used to distinguish individual molecules after sequencing that originated from unique mRNA molecules relative to PCR duplicates.
  • An index sequence can also be added that allows for multiplexing multiple libraries of barcodes on the same flow-cell or lane of a flow cell.
  • the resulting amplicon contains 5’ and 3’ sequencing primers and an index, the genetic perturbation, the barcode, an additional barcode (CB), and a unique molecular identifier (UMI).
  • a poly-A and poly-C tail are appended to the transcribed RNA (FIG. 1C).
  • the construct contains a capture sequence (denoted as “CS”), either upstream, downstream, or both upstream and downstream of the barcode and the genetic perturbation.
  • Primers for amplification of the transcript are introduced and bind to the CS to convert the RNA into cDNA.
  • the primers are associated with the 5’ and 3’ ends of the nucleic acid for next generation sequencing.
  • the primers can also include an additional barcode (e.g., a cellular barcode (CB)), a unique molecular identifier sequence (UMI) and/or an index sequence.
  • the resulting amplicon contains 5’ and 3’ sequencing primers and index, the genetic perturbation, the barcode, a cellular barcode (CB), and a UMI.
  • the construct contains an artificial poly-A sequence inserted downstream of the barcode and/or agent (FIG. ID).
  • the artificial poly-A sequence mimics the poly-A tail appended during transcription.
  • Primers for amplification of the transcript bind to the poly-C tail and the artificial poly-A sequence to convert the RNA into DNA.
  • the primers are associated with 5’ and 3’ end of the nucleic acid for next generation sequencing and can be associated with a CB, a UMI and/or and index.
  • the resulting amplicon contains 5’ and 3’ sequencing primers, the genetic perturbation, the barcode, an additional cellular barcode (CB), a UMI, and an index.
  • the barcode is incorporated into DNA by genomic integration (e.g., vector or viral vector delivery) using the DNA integration method can also be used to track different cellular lineages or clonal cells.
  • the barcode is a marker for clonal cells.
  • the barcode is called a clonal barcode.
  • the clonal barcode is incorporated in such a way that an RNA transcript is produced carrying the clonal barcode.
  • the barcode can also be incorporated by a self-replicating mRNA molecule such that integration is not necessary, as long as it can be passed to the progeny. Incorporation of the clonal barcode ensures that only one clonal barcode is integrated per cell, although a combination of clonal barcodes can also be used to mark a unique group of cells.
  • the barcode allows the analysis to combine single cell data from the same clones. For example, statistical methods are used to determine numerous weak signals from many single cells (from the same clone) to give a “strong” signal when pseudo bulked together. This is useful for calling of copy numbers or single nucleotide variants, which are challenging to estimate from current single cell sequencing methods that are inaccurate and costly. The methods provided herein require fewer data points per single cell which also saves costs.
  • Single cell sequencing method When performing single-cell sequencing, due to the stochastic nature of RNA (or protein/nucleic acid) capture, approximately 2000-4000 genes are captured, which account for only 20% of transcriptome. Detection of genetic alterations and perturbations (e.g., point mutations, copy number alterations, insertions and deletions, etc.) from single-cell RNA-sequencing data by traditional sequencing methods is poor and expensive. Thus, in traditional single-cell analysis of cell/clonal outgrowth a mutation may be detected in a small percentage of cells (e.g., 10%), but it is unknown if this is representative of the true population of cells comprising the mutation (i.e.
  • the methods provided herein track single cells back to their original starting populations, information that can then be used to group cells by their starting population, forming pseudo bulk populations.
  • Single cells within these pseudo bulk populations can be used to define the characteristics of the entire population. For example, if a mutation is detected in one or more single cells (but not all) from a particular pseudo bulk population, it can be estimated that all the cells within the subpopulation have the alteration. In other words, this approach enables label propagation of genomic alteration calls to capture the true heterogeneity of a cell population.
  • Genetic perturbation method A challenge with traditional single-cell RNA sequencing is that a population of cells with the same genetic alteration/perturbation in a heterogenous cell line, traditional sequencing captures a mixture of cellular clones and not a specific subpopulation. This limitation impacts the ability to identify a true phenotype or a clonal outgrowth.
  • the methods provided herein enable deconvolution of the genetically modified population and refine true clonal phenotype. As a result, the genetic perturbation-induced phenotype is unique to a specific clone, unique to specific subpopulations of cells, or is common to all cells within the tested population.
  • FIGS. 2A-2B and FIGS. 3A-3B Exemplary methods of clonal barcoding are illustrated in FIGS. 2A-2B and FIGS. 3A-3B.
  • a unique molecular identifier (UMI) or a cellular barcode (CB) is introduced to a target nucleic acid with a genetic perturbation.
  • Cells are grown to allow for clonal outgrowth, and single-cell analysis is performed. This captures the barcode, the genetic perturbation identifier (showing which genetic perturbation has occurred) and the genetic landscape simultaneously. For analysis, cells can then be grouped together if they have the same barcode as this indicates the original starting cell. This allows the impact of the genetic perturbation to be linked back to the original cell clone.
  • the analysis also increases confidence in the data as the methods provided herein make it possible to identify genetic perturbations and/or genetic alterations that impact multiple clones rather than just a single clone.
  • Having an accurate representation and genomic characterization of molecular tumor sub-populations enables identification of genetic features associated with a differential response to gene editing or an agent.
  • cells are transduced with nontargeting guide RNA or guide RNA that specifically binds to a given gene for genetic perturbation by a gene editing system (e.g., a Cas enzyme).
  • the gene expression changes in response to genetic perturbation of gene X can be determined by calculating differential expression of genetically altered cells compared to population of cells carrying non-targeting guide RNA. Since cells are labelled both by individual cell clone and by a respective sub-population, it becomes possible to study differential transcriptional (FIG. 4B) and phenotypic (FIG. 4C) responses between tumor sub-populations.
  • Gene editing screening of heterogeneous tumor cell-lines enables the association of differential responses or phenotypes to sets of genetic features by combining feature selection methods (e.g., mutual information, recursive feature elimination, Lasso regression, Boruta algorithm etc.) and machine learning models such as random forest and gradient boosting.
  • feature selection methods e.g., mutual information, recursive feature elimination, Lasso regression, Boruta algorithm etc.
  • machine learning models such as random forest and gradient boosting.
  • the methods provided herein allow for the pairing of a transcriptional response (e.g., tumor sternness) or cellular phenotype e.g., cell death) and cancer-associated genetic features with much greater resolution and accuracy. This approach can yield new targets for drug discovery applications.
  • Example 3 Application for Target Discovery in Single Cell.
  • dCas9 glioblastoma multiforme (GBM) cells lines were generated via transduction of the cells with lentiviruses expressing dCas9 KRAB at a multiplicity of infection (MOI) of 1 in the presence of polybrene (5 pg/ml).
  • MOI multiplicity of infection
  • dCas9 KRAB was constitutively expressed under an EFla promoter.
  • Puromycin was used to select for transduced cells. Three days after transduction, cells were selected for one week using puromycin (5 pg/ml).
  • the cells were routinely maintained in NeuroCultTM Proliferation Medium [Human] supplemented with recombinant human EGF (20 pg/ml), recombinant human bFGF (20 pg/ml) and heparin (2 pg/ml).
  • scRNA-seq screen A lentiviral vector used for these studies contained two main components: an EFla promoter driving the constitutive expression of EGFP, and a U6 promoter driving the constitutive expression of the gRNA library.
  • the EFla promoter also drives the expression of the poly-adenylated gRNA which is used for gRNA capture.
  • the gRNA backbone downstream of the gRNA contained the UMT consisting of 6 degenerate nucleotides (6N).
  • dCas9 GBM cell lines were infected with a sgRNA lentiviral library harboring 1620 sgRNAs associated with UMTs targeting 450 human genes at an MOI of 0.5 in the presence of polybrene (5 pg/ml). During the whole screen, cell numbers were maintained at a library coverage of at least 300 cells per sgRNA using flow for GFP at each split of the cells to determine the number of sgRNA-positive cells.
  • a lentiviral dCas9 plasmid was generated as described in Example 3.
  • Generation of dCas9 cell lines dCas9 glioblastoma multiforme (GBM) cells lines were generated via transduction of the cells with lentiviruses expressing dCas9 KRAB at a multiplicity of infection (MOI) of 1 in the presence of polybrene (5 pg/ml). Three days after transduction, cells were selected for one week using puromycin (5 pg/ml).
  • the cells were routinely maintained in NeuroCult Proliferation Medium [Human] supplemented with recombinant human EGF (20 pg/ml), recombinant human bFGF (20 pg/ml) and heparin (2 pg/ml).
  • Pooled dropout screen dCas9 GBM cell lines were infected with the same sgRNA lentiviral library as for the scRNA-seq screen at an MOI of 0.5 in the presence of polybrene (5 pg/ml) and at a minimum of 300-fold library coverage.
  • a first step 5 pg of gDNA per reaction was amplified with 14 PCR cycles using Q5 Hot Start High-Fidelity DNA Polymerase (NEB MO491L) and a set of 4 tiling forward primers and one reverse primer to increase library complexity.
  • Forward primers were designed to bind upstream of the sgRNA (U6 promoter), and the reverse primer was designed to bind to a constant region downstream of the UMT (sgRNA backbone).
  • PCR reactions were cleaned up using SPRI beads (Cat M1378-01) and eluted in 30.5 pl of EB buffer.
  • PCR product 30 pl was subjected to a second round of PCR using 12 cycles of PCR with primers that add Illumina i5 and i7 sequences as well as indexing barcodes for multiplexing downstream of the UMT.
  • the final PCR product was purified using SPRI beads and sequenced using single-end (SE) sequencing (lOObp, i7 8bp) on Illumina NextseqTM 2000 at 40 million reads per library.
  • 2FAST2( ⁇ (PMID. 36312750) was used to extract and count the expected amplicon, where amplicon here refers to the sgRNA, the sgRNA backbone and the UMT (FIG. 5).
  • amplicon here refers to the sgRNA, the sgRNA backbone and the UMT (FIG. 5).
  • 2FAST2Q “extract and count mode”, which retrieves any sequences between user-provided upstream and downstream constant search sequences; in our case, these constant search sequences corresponded to the 10 bp upstream of the sgRNA and 10 bp downstream of the UMT.
  • a minimal Phred-score of Q 3 10 was required across (i) both search sequences and (ii) the extracted sequence.
  • sequences extracted by 2FAST2Q were filtered using custom code to include only those of the expected amplicon length, which here equated to 45 bp (20 bp sgRNA + 19 bp backbone + 6 bp UMT). Thereafter, sgRNA sequences and UMTs were extracted using custom code based on their expected position in the amplicon and their length. Here, the sgRNA is expected to make up the first 20 elements in the amplicon sequence, while the UMT sequence would make up the last 6 elements. Once sgRNA and UMT sequences were extracted, counts were summed across each distinct combination of sgRNA and UMT.

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Abstract

L'invention concerne des procédés d'identification d'une altération génétique dans une séquence d'acide nucléique par l'intermédiaire de compositions de marquage moléculaire. Des procédés, des compositions et des kits selon l'invention peuvent être utilisés en tant qu'outils biotechnologiques pour analyser des interactions génétiques, un phénotypage cellulaire, une génomique unicellulaire et des cribles de système d'édition génique.
PCT/IB2024/057230 2023-07-25 2024-07-25 Procédés de codage à barres de cellules uniques et de sous-populations cellulaires et leurs utilisations Pending WO2025022355A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210189352A1 (en) * 2017-10-13 2021-06-24 Imba - Institut Für Molekulare Biotechnologie Gmbh Enhanced reprogramming of somatic cells
US20220259593A1 (en) * 2019-07-26 2022-08-18 The Regents Of The University Of California Control of mammalian gene dosage using crispr
US20220392569A1 (en) * 2021-05-26 2022-12-08 Industry-Academic Cooperation Foundation, Yonsei University Method for evaluating the function of cancer mutations through base editor and evaluation system using the same
US20230121309A1 (en) * 2020-03-30 2023-04-20 Imba - Institut Für Molekulare Biotechnologie Gmbh Internal standard for crispr guide rna

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210189352A1 (en) * 2017-10-13 2021-06-24 Imba - Institut Für Molekulare Biotechnologie Gmbh Enhanced reprogramming of somatic cells
US20220259593A1 (en) * 2019-07-26 2022-08-18 The Regents Of The University Of California Control of mammalian gene dosage using crispr
US20230121309A1 (en) * 2020-03-30 2023-04-20 Imba - Institut Für Molekulare Biotechnologie Gmbh Internal standard for crispr guide rna
US20220392569A1 (en) * 2021-05-26 2022-12-08 Industry-Academic Cooperation Foundation, Yonsei University Method for evaluating the function of cancer mutations through base editor and evaluation system using the same

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LI CHONG ET AL: "Single-cell brain organoid screening identifies developmental defects in autism", BIORXIV, 15 September 2022 (2022-09-15), pages 1 - 26, XP093220610, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2022.09.15.508118v1.full.pdf> [retrieved on 20241106], DOI: 10.1101/2022.09.15.508118 *
MICHELS BIRGITTA E. ET AL: "Pooled In?Vitro and In?Vivo CRISPR-Cas9 Screening Identifies Tumor Suppressors in Human Colon Organoids", CELL STEM CELL, vol. 26, no. 5, 7 May 2020 (2020-05-07), AMSTERDAM, NL, pages 782 - 792.e7, XP093220120, ISSN: 1934-5909, DOI: 10.1016/j.stem.2020.04.003 *
ZHU SHIYOU ET AL: "Guide RNAs with embedded barcodes boost CRISPR-pooled screens", GENOME BIOLOGY, vol. 20, no. 20, 24 January 2019 (2019-01-24), pages 1 - 12, XP055968304, DOI: 10.1186/s13059-019-1628-0 *

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