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WO2025137335A1 - Imagerie optique d'acides nucléiques - Google Patents

Imagerie optique d'acides nucléiques Download PDF

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Publication number
WO2025137335A1
WO2025137335A1 PCT/US2024/061106 US2024061106W WO2025137335A1 WO 2025137335 A1 WO2025137335 A1 WO 2025137335A1 US 2024061106 W US2024061106 W US 2024061106W WO 2025137335 A1 WO2025137335 A1 WO 2025137335A1
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Prior art keywords
promoter
nucleic acid
cells
certain embodiments
nucleotide sequence
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Eric Scott LUBECK
Takamasa Kudo
Aviv Regev
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Genentech Inc
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Genentech Inc
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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
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/127—RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
    • C12N9/226—Class 2 CAS enzyme complex, e.g. single CAS protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • the present disclosure relates to methods for optically imaging nucleic acids in single cells and compositions for use in the disclosed methods.
  • nucleic acid expression and distribution is useful in understanding complex biological systems.
  • the analysis of nucleic acid expression has facilitated the identification of genes involved in organismal growth and development, as well as the determining the causes and analyzing the progression of a wide variety of diseases.
  • Conventional strategies for detecting and characterizing nucleic acids within the cellular context comprise in situ hybridization and in situ sequencing. These strategies, however, do not allow for the detection of nucleic acids in different cell types in complex biological systems to facilitate the identification of cellular phenotypes associated with the expression of such nucleic acids. Accordingly, there remains a need in the art for improved methods for the analysis of nucleic acids, e.g., across different cell types.
  • a method of the present disclosure comprises (a) providing a plurality of cells, wherein at least one cell of the plurality of cells comprises a nucleic acid construct comprising a first promoter, a second promoter and a target nucleic acid, wherein the first promoter and the second promoter are located upstream to the target nucleic acid in the nucleic acid construct, (b) culturing the plurality of cells to allow expression of the target nucleic acid using the first promoter, (c) fixing the plurality of cells to generate a plurality of fixed cells, (d) transcribing the target nucleic acid in the at least one cell of the plurality of fixed cells using the second promoter, (e) performing an amplification process to amplify the target nucleic acid and (f) imaging the amplified target nucleic acid in the at least one cell.
  • the target nucleic acid comprises from about 4 to about 1,000 nucleotides.
  • the target nucleic acid encodes a guide RNA (gRNA), a microRNA (miRNA), a small nucleolar RNA (snoRNA), a small interfering RNA (siRNA), piwi-interacting RNAs (piRNAs), aptamers, ribozymes, endogenous siRNAs (endo-siRNAs), a short hairpin RNA (shRNA) or a combination thereof.
  • the target nucleic acid encodes a gRNA.
  • the gRNA has an editing efficiency greater than about 60%, e.g., an editing efficiency greater than about 80% or an editing efficiency greater than about 90%.
  • the present disclosure further provides a method for performing a genomic screen that comprises (a) providing a plurality of cells, wherein at least one cell of the plurality of cells comprises a nucleic acid construct comprising a first promoter, a second promoter and a target nucleic acid encoding a gRNA, wherein the first promoter and the second promoter are located upstream to the target nucleic acid in the nucleic acid construct, (b) culturing the plurality of cells to allow expression of the gRNA using the first promoter, (c) fixing the plurality of cells to generate a plurality of fixed cells, (d) transcribing the gRNA in at least one cell of the plurality of fixed cells using the second promoter, (e) performing an amplification process for amplifying the gRNA; (f) imaging the amplified gRNA in the at least one cell and (g) analyzing a change in a characteristic of the at least one cell of the plurality of cells associated with expression of the gRNA.
  • the plurality of cells is decrosslinked prior to performing the amplification process.
  • the amplified target nucleic acids are imaged by in situ sequencing. In certain embodiments, the amplified target nucleic acids are imaged by fluorescent in situ hybridization. In certain embodiments, the nucleic acid construct further comprises a polynucleotide encoding a nuclease. In certain embodiments, the nucleic acid construct further comprises a barcode. In certain embodiments, the barcode is located downstream of the target nucleic acid.
  • the plurality of cells comprises a second cell that comprises a second nucleic acid construct comprising the first promoter, the second promoter and a second target nucleic acid encoding a second gRNA, wherein the first promoter and the second promoter are located upstream to the second target nucleic acid in the second nucleic acid construct.
  • a method of the present disclosure further comprises analyzing a change in a characteristic of one or more cells in the plurality of cells expressing the target nucleic acid compared to one or more cells in the plurality of cells that does not express the target nucleic acid. In certain embodiments, analyzing a change in a characteristic of one or more cells is performed prior to imaging the amplified target nucleic acid in the at least one cell. In certain embodiments, analyzing a change in a characteristic of one or more cells in the plurality of cells expressing the target nucleic acid is performed after fixation of the plurality of cells and prior to transcription of the target nucleic acid, e.g., using the second promoter of the hybrid promoter. In certain embodiments, analyzing a change in a characteristic of one or more cells is performed after fixation but prior to decrosslinking.
  • the characteristic is selected from the group consisting of cell viability, cell proliferation, cell size, cell morphology, cell motility, cell differentiation, cell adhesion, cell-cell contact, mutational status, karyotype, chromosomal aberrations, nucleic acid expression levels (e.g., mRNA expression levels), protein expression levels, nucleic acid modifications (e.g., methylation), post-translational modifications (e.g., phosphorylation, ubiquitination and/or glycosylation), activation of a cell signaling pathway, repression of a cell signaling pathway, enzymatic activity (e.g., enzymatic cleavage), chromatin accessibility, histone modifications and other epigenetic changes, concentrations of cytokines and hormones, drug sensitivity, drug absorption and metabolism pharmacokinetics and pharmacodynamics, membrane potential and a combination thereof.
  • the characteristic is protein expression levels.
  • the characteristic is protein expression localization.
  • a method of the present disclosure further comprises performing an immunofluorescence process for detecting one or more target proteins, e.g., performing an immunofluorescence process for detecting one or more, two or more, three or more, four or more or five or more target proteins.
  • the immunofluorescence process for detecting the one or more target proteins is performed after fixation of the plurality of cells and prior to transcription of the target nucleic acid, e.g., using the second promoter of the hybrid promoter.
  • the immunofluorescence process for detecting the one or more target proteins is performed after fixation but prior to decrosslinking.
  • kits for performing a method described herein comprises at least one container comprising a nucleic acid construct comprising a first promoter, a second promoter and a target nucleic acid, wherein the first promoter and the second promoter are located upstream to the target nucleic acid in the nucleic acid construct.
  • the nucleotide sequence of the second promoter is incorporated into the nucleotide sequence of the first promoter.
  • the nucleotide sequence of the second promoter is incorporated into nucleotides 40 to about 200 located downstream from the 5’ end of the nucleotide sequence of the first promoter.
  • the first promoter is a promoter for expression in live cells. In certain embodiments, the first promoter is a Pol III or a Pol II promoter. In certain embodiments, the first promoter is a Pol III promoter. In certain embodiments, the first promoter is a Pol II promoter. In certain embodiments, the first promoter is selected from the group consisting of a U6 promoter, U3 promoter, U2 promoter, U5 promoter, Hl promoter, 7SK promoter, 75J promoter, EF-la promoter, CMV promoter, a tRNA promoter, pGK promoter, SV40 promoter, CAG promoter, TRE promoter, VAI promoter and a combination thereof.
  • the first promoter is a U6 promoter.
  • the second promoter is a promoter for expression in fixed cells.
  • the second promoter is a promoter for a phage RNA polymerase.
  • the phage promoter is selected from the group consisting of a T3 promoter, a T7 promoter, a Sp6 promoter and a combination thereof.
  • the phage promoter is a T7 promoter.
  • the nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 1-18.
  • the target nucleic acid in a nucleic acid construct of a kit of the present disclosure comprises from about 4 to about 1,000 nucleotides.
  • the target nucleic acid encodes a guide RNA (gRNA), a microRNA (miRNA), a small nucleolar RNA (snoRNA), a small interfering RNA (siRNA), piwi- interacting RNAs (piRNAs), aptamers, ribozymes, endogenous siRNAs (endo-siRNAs), a short hairpin RNA (shRNA) or a combination thereof.
  • the target nucleic acid encodes a gRNA.
  • the nucleic acid construct further comprises a polynucleotide encoding a nuclease.
  • the nucleic acid construct further comprises a barcode.
  • kits of the present disclosure can further include a reducing agent, e.g., DTT.
  • a kit of the present disclosure can include a reducing agent, e.g., DTT, in a container and/or within a buffer in a container.
  • the present disclosure further provides a nucleic acid construct comprising a first promoter comprising a first nucleotide sequence, a second promoter comprising a second nucleotide sequence and a target nucleic acid, wherein the nucleotide sequence of the second promoter is incorporated into the nucleotide sequence of the first promoter.
  • the first promoter and the second promoter are located upstream to the target nucleic acid in the nucleic acid construct.
  • the first promoter is a promoter for expression in live cells.
  • the first promoter is a Pol III promoter or a Pol II promoter.
  • the first promoter is selected from the group consisting of a U6 promoter, U3 promoter, U2 promoter, U5 promoter, Hl promoter, 7SK promoter, 75 J promoter, EF- la promoter, CMV promoter, a tRNA promoter, pGK promoter, SV40 promoter, CAG promoter, TRE promoter, VAI promoter and a combination thereof.
  • the first promoter is a U6 promoter.
  • the second promoter is a promoter for expression in fixed cells.
  • the second promoter is a promoter for a phage RNA polymerase.
  • the phage promoter is selected from the group consisting of a T3 promoter, a T7 promoter, a Sp6 promoter or a combination thereof. In certain embodiments, the phage promoter is a T7 promoter.
  • the nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 1-18.
  • the target nucleic acid comprises from about 4 to about 1,000 nucleotides.
  • the target nucleic acid encodes a guide RNA (gRNA), a microRNA (miRNA), a small nucleolar RNA (snoRNA), a small interfering RNA (siRNA), piwi-interacting RNAs (piRNAs), aptamers, ribozymes, endogenous siRNAs (endo-siRNAs), a short hairpin RNA (shRNA) or a combination thereof.
  • the target nucleic acid encodes a gRNA.
  • the gRNA has an editing efficiency greater than about 60%, e.g., an editing efficiency greater than about 80% or an editing efficiency greater than about 90%.
  • the nucleic acid construct further comprises a polynucleotide encoding a nuclease.
  • the nucleic acid construct further comprises a barcode.
  • the barcode is located downstream of the target nucleic acid (e.g., 3’ to the target nucleic acid).
  • the present disclosure further discloses a nucleic acid comprising a first promoter comprising a first nucleotide sequence, a second promoter comprising a second nucleotide sequence, wherein the nucleotide sequence of the second promoter is incorporated into the nucleotide sequence of the first promoter, wherein the nucleic acid comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 7-18.
  • the nucleic acid comprises the nucleotide sequence of any one of SEQ ID NOs: 7-18.
  • the present disclosure further provides a composition comprising a nucleic acid construct disclosed herein.
  • the present disclosure further provides a method for imaging a target nucleic acid in a plurality of cells that includes (a) providing a plurality of cells, wherein at least one cell of the plurality of cells comprises a nucleic acid construct comprising (i) a target nucleic acid and (ii) a hybrid promoter located upstream to the target nucleic acid in the nucleic acid construct, wherein the hybrid promoter comprises a first promoter and a second promoter, wherein the nucleotide sequence of the second promoter is incorporated into the nucleotide sequence of the first promoter, (b) culturing the plurality of cells to allow expression of the target nucleic acid using the first promoter of the hybrid promoter, (c) fixing the plurality of cells to generate a plurality of fixed cells, (d) transcribing the target nucleic acid in the at least one cell of the plurality of fixed cells using the second promoter of the hybrid promoter, (e) performing an amplification process to amplify the target nucle
  • imaging the amplified target nucleic acid comprises imaging the target nucleic acid by in situ sequencing.
  • the method further comprises analyzing a change in a characteristic of one or more cells in the plurality of cells expressing the target nucleic acid compared to one or more cells in the plurality of cells that does not express the target nucleic acid.
  • the characteristic is selected from the group consisting of cell viability, cell proliferation, cell size, cell morphology, cell motility, cell differentiation, cell adhesion, cell-cell contact, mutational status, karyotype, chromosomal aberrations, nucleic acid expression levels, nucleic acid localization, protein expression levels, protein localization, nucleic acid modifications, post-translational modifications, activation of a cell signaling pathway, repression of a cell signaling pathway, enzymatic activity, chromatin accessibility, histone modifications and other epigenetic changes, concentrations of cytokines and hormones, drug sensitivity, drug absorption and metabolism pharmacokinetics and pharmacodynamics, membrane potential and a combination thereof.
  • analyzing a change in a characteristic of one or more cells is performed prior to imaging the amplified target nucleic acid (e.g., by in situ sequencing) in the at least one cell.
  • the characteristic is protein expression levels.
  • the characteristic is nucleic acid expression levels.
  • the method further includes performing an immunofluorescence process for detecting one or more target proteins, e.g., one or more target proteins in one or more cells of the plurality of cells.
  • analyzing a change in a characteristic of one or more cells is performed prior to imaging the amplified target nucleic acid in the at least one cell.
  • analyzing a change in a characteristic of one or more cells in the plurality of cells expressing the target nucleic acid is performed after fixation of the plurality of cells and prior to transcription of the target nucleic acid, e.g., using the second promoter of the hybrid promoter.
  • the immunofluorescence process for detecting the one or more target proteins is performed after fixation of the plurality of cells and prior to transcription of the target nucleic acid, e.g., using the second promoter of the hybrid promoter.
  • analyzing a change in a characteristic of one or more cells is performed after fixation but prior to decrosslinking.
  • the present disclosure further provides a method for imaging a target nucleic acid in a plurality of cells, comprising: (a) providing a plurality of cells, wherein at least one cell of the plurality of cells comprises a nucleic acid construct comprising a first promoter, a second promoter and a target nucleic acid, wherein the first promoter and the second promoter are located upstream to the target nucleic acid in the nucleic acid construct, (b) culturing the plurality of cells to allow expression of the target nucleic acid using the first promoter, (c) fixing the plurality of cells in a fixative comprising aldehyde to generate a plurality of fixed cells, (d) decrosslinking the plurality of fixed cells to generate a plurality of decrosslinked cells, (e) transcribing the target nucleic acid in the at least one cell of the plurality of decrosslinked cells using the second promoter of the hybrid promoter, (f) performing an amplification process to amplify the target nucleic acid
  • imaging the amplified target nucleic acid comprises imaging the target nucleic acid by in situ sequencing.
  • the method further comprises analyzing a change in a characteristic of one or more cells in the plurality of cells expressing the target nucleic acid compared to one or more cells in the plurality of cells that does not express the target nucleic acid.
  • the characteristic is selected from the group consisting of cell viability, cell proliferation, cell size, cell morphology, cell motility, cell differentiation, cell adhesion, cell-cell contact, mutational status, karyotype, chromosomal aberrations, nucleic acid expression levels, nucleic acid localization, protein expression levels, protein localization, nucleic acid modifications, post-translational modifications, activation of a cell signaling pathway, repression of a cell signaling pathway, enzymatic activity, chromatin accessibility, histone modifications and other epigenetic changes, concentrations of cytokines and hormones, drug sensitivity, drug absorption and metabolism pharmacokinetics and pharmacodynamics, membrane potential and a combination thereof.
  • analyzing a change in a characteristic of one or more cells is performed prior to imaging the amplified target nucleic acid in the at least one cell. In certain embodiments, analyzing a change in a characteristic of one or more cells in the plurality of cells expressing the target nucleic acid is performed after fixation of the plurality of cells and prior to transcription of the target nucleic acid, e.g., using the second promoter of the hybrid promoter. In certain embodiments, analyzing a change in a characteristic of one or more cells is performed after fixation but prior to decrosslinking. In certain embodiments, analyzing a change in a characteristic of one or more cells is performed after fixation but prior to decrosslinking. In certain embodiments, the characteristic is protein expression levels.
  • the characteristic is nucleic acid expression levels.
  • the method further includes performing an immunofluorescence process for detecting one or more target proteins, e.g., one or more target proteins in one or more cells of the plurality of cells.
  • the immunofluorescence process for detecting one or more target proteins is performed after permeabilization of the plurality of cells and prior to transcription of the target nucleic acid using the second promoter of the hybrid promoter.
  • the immunofluorescence process for detecting one or more target proteins is performed after fixation but prior to decrosslinking.
  • the present disclosure further provides a method for analyzing one or more characteristics of a plurality of cells.
  • the method includes (a) providing a plurality of cells, wherein at least one cell of the plurality of cells comprises a nucleic acid construct comprising a first promoter, a second promoter and a target nucleic acid, and wherein the first promoter and the second promoter are located upstream to the target nucleic acid in the nucleic acid construct, (b) culturing the plurality of cells to allow expression of the target nucleic acid using the first promoter of the hybrid promoter, (c) fixing the plurality of cells to generate a plurality of fixed cells, (d) detecting one or more characteristics of the at least one cell of the plurality of fixed cells, (e) transcribing the target nucleic acid in the at least one cell of the plurality of fixed cells using the second promoter of the hybrid promoter, (f) performing an amplification process to amplify the target nucleic acid, (g) imaging the ampl
  • the characteristic is selected from the group consisting of cell viability, cell proliferation, cell size, cell morphology, cell motility, cell differentiation, cell adhesion, cell-cell contact, mutational status, karyotype, chromosomal aberrations, nucleic acid expression levels, nucleic acid localization, protein expression levels, protein localization, nucleic acid modifications, post-translational modifications, activation of a cell signaling pathway, repression of a cell signaling pathway, enzymatic activity, chromatin accessibility, histone modifications and other epigenetic changes, concentrations of cytokines and hormones, drug sensitivity, drug absorption and metabolism pharmacokinetics and pharmacodynamics, membrane potential and a combination thereof.
  • the method further includes decrosslinking the plurality of cells prior to performing the amplification process.
  • the nucleotide sequence of the second promoter is incorporated into the nucleotide sequence of the first promoter.
  • the present disclosure further provides systems for performing the methods disclosed herein.
  • FIG. 1 provides a schematic of an exemplary method according to the present disclosure that can comprise fixation and permeabilization of cells followed by decrosslinking.
  • the exemplary method can further comprise performing T7 in vitro transcription followed by in situ sequencing to image the target nucleic acid.
  • in situ sequencing can comprise performing a reverse transcription process, a gap filling process, rolling circle amplification (RCA) and sequencing by synthesis.
  • FIG. 2 provides immunofluorescence (Left panel: bottom left, Right panel: Top Left), DAPI staining (Left panel: Top Left, Right panel: Bottom left) and in situ sequencing images of a nucleic acid in A549 cells and neurons using a previously known in situ sequencing technique. Bases sequenced are specified in the inset text on the in situ sequencing figures.
  • FIG. 3A provides a schematic of an exemplary nucleic acid construct according to the present disclosure.
  • FIG. 3B provides exemplary promoter sequences for use in the nucleic acid constructs of the present disclosure.
  • FIG. 4 shows that the incorporation of the T7 promoter into the U6 promoter does not affect the editing efficiency of the gRNAs under control of the U6 promoter.
  • U6 ,r/ refers to the wild type form of the U6 promoter and U6 r/ , U6 V2 , U6 V3 , U6 V4 and U6 1 corresponds to the T7-U6 promoters shown in FIG. 3B.
  • FIG. 5A provides fluorescence in situ hybridization images showing that the incorporation of the T7 promoter into the U6 promoter resulted in increased expression of gRNAs that can be detected by in situ sequencing (ISS) in A549 cells compared to the standard ISS technique after T7 addition.
  • ISS in situ sequencing
  • Diagonal division in panel corresponds to a higher display contrast on the bottom right quadrant for the same image.
  • Left and right images are contrasted identically.
  • FIG. 5B provides fluorescence in situ hybridization images showing that the incorporation of the T7 promoter into the U6 promoter resulted in increased expression of gRNAs transcripts after T7 in vitro transcription in A549 cells.
  • FIG. 6 provides a schematic of an exemplary screen in primary cells using the presently disclosed methods.
  • FIG. 7 shows that many NFKB pathway components were identified using the screen shown in FIG. 6.
  • FIG. 8 provides a schematic of an exemplary screen using the presently disclosed methods.
  • FIG. 9A provides an overview of an exemplary method of the present disclosure.
  • FIGS. 9B-9C show the CRISPR editing efficiency (FIG. 9B) and in vitro transcription activity (FIG. 9C).
  • Top a standard CROPseq Target 1 sgRNA (gray).
  • Asterisks chosen Perturb View construct.
  • FIG. 9D illustrates that Perturb View enables bright in vitro transcription from the U6 promoter with minimal modifications to the U6 sequence.
  • Top WT U6 promoter and Perturb View construct sequences.
  • FIG. 9E provides a frame-shift reporter screen to determine sensitivity and specificity of barcode detection.
  • Left Representative image of cells with immunofluorescence (IF) of HA-staining (right) or guide decoding with screen assignments (left, TP, FN and TN labeled by presence or absence of HA epitope and the first base of reads) (scale bar, 25 pm).
  • IF immunofluorescence
  • HA-staining right
  • guide decoding with screen assignments left, TP, FN and TN labeled by presence or absence of HA epitope and the first base of reads
  • FIG. 9H shows that Perturb View performs well across primary and iPSC-derived cells.
  • Left Representative images of sgRNA barcode detection by conventional ISS and Perturb View across cell types (Scale bar, 25 pm). All images are acquired identically except images denoted by an asterisk (* denotes a four-fold longer exposure duration to account for the drastically dimmer signal of some cell types in standard ISS experiments).
  • Right Fraction of cells positive for sgRNA signal (>1 sgRNA read per cell, mean of well replicates with error bar indicating SD, each dot representing a replicate) across cell types.
  • FIG. 10A shows the varied activity of the CROP-Seq vector by HCR FISH in primary cells.
  • HCR FISH of CROP-Seq expressed transcript green; detected with FISH probes against puromycin and U6 promoter regions; Table 1C) across different cell lines (scale bar, 200 pm).
  • CROP-Seq transcript density in A549 (left) and mouse BMDM (right) cells scale bar, 50 pm). All cells were transduced at low MOI and puromycin-selected.
  • FIG. 10B highlights that the position of the T7 promoter dramatically affects IVT- enhanced ISS signal.
  • Bottom, representative cellular images from each construct (scale bar, 25 pm).
  • FIG. 10D shows that the editing efficiency of the Perturb View vector is identical to wild-type U6 promoter (U6WT) in A549 cells, immortalized macrophages and fibroblasts.
  • U6WT wild-type U6 promoter
  • FIGS. 10E-10F show that de-crosslinking and IVT duration impact sensitivity and precision of barcode detection.
  • Error bars SD. Top, representative cellular images from each condition (scale bar, 25 pm).
  • FIG. 10G shows that Perturb View enables in situ sequencing at 4x magnification. Left, whole well (top; scale bar, 1 mm) or zoomed image (bottom; scale bar, 50 pm) representative image acquired at 4X magnification with either conventional ISS (left) or Perturb View (right). All images are contrasted identically. Heat decrosslinking prior to Perturb View results in increased nuclear DAPI intensity.
  • FIG. 101 shows the mean sensitivity (blue) and precision (orange) with (65°C) or without (25°C) decrosslinking in methanol-fixed cells (left) or PFA-fixed cells (right).
  • FIG. 10J shows that DTT rescues IVT efficiency at low T7 polymerase concentration in MCF7 cells.
  • Top representative cellular images from each condition (scale bar, 25 pm).
  • Bottom Population mean of the maximum normalized intensity of in situ sequencing spots. Each dot represents technical replicates (n > 2). Error bars: SD.
  • FIG. HA provides an overview of an exemplary NFKB screen in primary mouse BMDMs.
  • FIG. 11B illustrates that Perturb View but not conventional OPS sensitively detects sgRNA barcodes in BMDMs.
  • Left Reads (fluorescent signals) from standard ISS (top) and Perturb View (bottom) in BMDMs (Scale bar, 25 pm).
  • FIGS. 11C-11E provides the shared and context-specific hits from Perturb View OPS in three stimuli. Significance (-logio(FDR), y axis) and effect size (normalized change in nuclear p65 intensity) for each perturbation (dot) in TNFa (FIG. 11C), IL- 1 P (FIG. 1 ID), and LPS (FIG. HE) stimuli, colored for positive (blue; FDR ⁇ 0.05), negative (orange; FDR ⁇ 0.05), control (red, non-targeting sgRNA and non-essential genes) perturbations.
  • Significance -logio(FDR), y axis) and effect size (normalized change in nuclear p65 intensity) for each perturbation (dot) in TNFa (FIG. 11C), IL- 1 P (FIG. 1 ID), and LPS (FIG. HE) stimuli, colored for positive (blue; FDR ⁇ 0.05), negative (orange; FDR ⁇
  • FIG. 11F provides an exemplary workflow for multimodal NFKB Perturb View screen using RNA FISH (HCR) and IF (IBEX). Representative image of BMDM assayed in order by HCR FISH (left), immunofluorescence (middle), and Perturb View (right).
  • FIG. 11G shows successful sgRNA barcode detection in multimodal Perturb View, but not conventional OPS.
  • Mean percent of cells with detected barcode (y axis) in conventional OPS, conventional OPS after FISH and IF, and multi-modal Perturb View (x axis).
  • Error bars SDXX. Dots: each well and condition.
  • FIGS. 11H-11I show co-functional perturbation modules based on multimodal Perturb View.
  • PHATE embedding of perturbation profiles (dots) following TNFa (FIG. 11H) or IL-ip (FIG. HI) stimulus sized by q value of perturbation effect and colored by cluster or as controls.
  • Gene labels are shown for q-value ⁇ 0.02 in selected clusters (1 and 6 in (FIG. 11H); 1, 2, 4 and 6 in (FIG. HI)).
  • FIG. 11 J provides a schematic of an exemplary workflow for a multimodal NFKB Perturb View screen using RNA FISH (HCR) and IF (IBEX).
  • FIG. 12A shows agreement in sgRNA detection in Perturb View and NGS.
  • FIG. 12B highlights the intersection of hit genes between TNFa, ILip, and LPS Perturb View screens (circles) of NFKB translocation in primary BMDMs.
  • CDFs cumulative distribution functions
  • FIG. 12F shows PHATE embeddings of RNA/protein joint profiles (dots) for each sgRNA in the TNFa (left) or ILip (right) screen targeting a gene (color) or controls (grey). Gene names are shown for each guide whose 5 nearest neighbors contains another guide targeting the same gene.
  • FIG. 12G shows the distribution of cosine similarity of the phenotypic profiles embeddings derived from joint RNA/protein profiling for random pairs of guides (“-”, blue) and either impactful guides targeting the same gene in the (orange; top) or targeting Olfir genes (orange bottom).
  • FIG. 12H shows the contribution of different molecular phenotypes to perturbation impact. Impact score (p-values UNIT, color bar) for each perturbed gene (row) in the TNFa (left), ILip (middle), or LPS (right) screen, when assessed only based on one imaging feature (RNA, protein or DAPI; columns). Rows and columns are clustered by hierarchical clustering.
  • FIG. 121 shows representative cellular images of p65 staining (top) and segmentation results (bottom) (Scale bar, 25 pm).
  • FIG. 12J shows that PerturbView enables efficient sgRNA recovery after multiplexed imaging techniques.
  • BMDMs were stained with F4/80 repeatedly using 4i, IBEX and cycIF (Scale Bar, 50 pm).
  • Middle Representative images of in situ sequencing results after 6 rounds of staining (Scale Bar, 50 pm).
  • FIG. 12K shows that CDFs of phospho-rpS6 intensity in response to TNFa for four guides (colored curves), compared to cells with non-targeting or non-essential controls (gray line (combined); shading standard deviation) (Top). FDRs were computed among the gene list in FIG. 12H. Representative phospho-rpS6 images under perturbations are shown (Bottom). Two cells were sampled from each of a 10-percentile group (according to the mean cellular phospho-rpS6 level) and arranged from low (left) to high (high).
  • FIG. 13E provides a tissue section image of Xenium spatial transcriptomics (left; 377 human pan-cancer markers) followed by sgRNA identification with PerturbView (right).
  • FIGS. 13F-13G provide a tumor section colored by sgRNA clone (FIG. 13F), Shannon index computed from clonal analysis (FIG. 13G, top) or low, middle and high Shannon diversity (FIG. 13G, bottom).
  • FIG. 131 shows a representative image of PRKDC immunofluorescence for staining human cells.
  • Mouse cells (outer rim) or necrotic regions (central region) show low PRKDC staining. Scale bar, 1 mm.
  • FIG. 13J shows spatial transcriptome and barcode detection in UMAP and spatial domains. From left to right: (1) UMAP based on transcriptome with each color and number represents a Leiden cluster at a resolution of 0.5; (2) corresponding Leiden clusters visualized in spatial domain; (3) Total transcript counts in each cell; (4) barcode’s minimum hamming distance in spatial domain (showing hamming distance up to 2).
  • FIG. 13K shows tumor sections profiled (scale bar, 1 mm), colored by Shannon diversity (top) or corresponding diversity groups (bottom) for all analyzed sections aside from the one shown in FIG. 13H.
  • FIG. 13M shows sgRNA detection efficiency in DLD-1 (PRKDC+) xenograft model.
  • Left representative image of DAPI (magenta), PRKDC (cyan) and nuclear segmentation (yellow) with (+) or without (-) detected sgRNAs. Scale bar, 50 pm.
  • FIG. 13N provides an exemplary workflow that include Perturb View and Xenium.
  • DLD-1 cells carrying a 100 NTC barcode library were implanted to a mouse xenograft model, followed by Xenium spatial transcriptomics, and in situ sequencing of sgRNAs with Perturb View.
  • Right UMAP embedding of single cell profiles (dots, top) or zoomed in tumor section (bottom, scale bar, 200 pm) from Xenium colored by Leiden clusters (left) or sgRNA identity (right).
  • FIG. 14B shows the Fraction of barcodes (x axis) mapped at different Hamming distances (colors) in each of four samples (y axis).
  • FIG. 14C shows the clonal aggregation of gene expression to enhance complexity in Xenium measurements. Distribution of number of unique genes detected (x axis) by Xenium in single cells or per clone (in aggregate) (x axis).
  • FIG. 14D shows the clonal maps and associated expression agree across serial sections. Clonal maps colored by Shannon diversity index of two consecutive sections (left and right) in each of two tumors (top and bottom).
  • FIG. 14E shows the genes (columns) that are differentially expressed between regions with high and low Shannon diversity (rows) for each of two consecutive tissue sections in each of two tumors (left vertical line indicates a pair of adjacent sections).
  • the present disclosure relates, in certain embodiments, to methods for the imaging of nucleic acids, e.g., gRNAs, expressed in sample, e.g., a sample comprising a plurality of cells.
  • the present disclosure further provides, in certain embodiments, nucleic acid constructs and other compositions for performing the disclosed methods.
  • methods of the present disclosure allow for the visualization of a target nucleic acid in a cell.
  • the cell is present within a plurality of cells.
  • method further comprises the correlation of the presence of the target nucleic acid to the characteristics of the cell. For example, but not by way of limitation, methods of the present disclosure can be used to determine phenotypic characteristics of cells present in a sample (e.g., in a plurality of cells) that express the target nucleic acid.
  • methods of the present disclosure can be used in screening methods.
  • methods of the present disclosure can be used in genomic screening, e.g., for performing optical genetic screens.
  • the detection of gRNA expression in a cell can be associated with the phenotype and/or genotype of the cell to determine the function of the gene targeted by the gRNA.
  • RNA barcodes As described herein, known methods of in situ sequencing have been shown to be inefficient for imaging nucleic acids in certain contexts, e.g., in complex biological systems such as primary cell lines and tissue.
  • the use of in situ sequencing is not compatible with many cell types, which can limit the use of in situ sequencing in biological systems that comprise a plurality of cell types.
  • the present disclosure provides methods for imaging target nucleic acids in a plurality of cell types, as shown in FIG. 4B, FIG. 9G and FIG. 13A.
  • methods of the present disclosure provide for the imaging of target nucleic acids, including target nucleic acids in complex biological systems.
  • complex biological systems can comprise a plurality of diverse cells such as those in situ.
  • the presently disclosed methods allow for in situ sequencing in a complex biological system such as a cancer xenograft model in combination with phenotypic analyses (e.g., analysis of gene expression).
  • phenotypic analyses e.g., analysis of gene expression.
  • the disclosed method enables complex genotype/phenotype investigations such as understanding modifications to therapeutically relevant cell types (e.g., T cells, neurons, macrophages, etc.) to better enable optimizations of these cell types for cell therapy applications.
  • the present disclosure is based, in part, on methods comprising the in vitro transcription of a target nucleic acid introduced into a cell, followed by amplification of the transcribed target nucleic acid, to allow for the robust visualization of the target nucleic acid in the cell via in situ sequencing.
  • the present disclosure is further based, in part, on the use of a hybrid promoter that allows amplification of a target nucleic acid in fixed cells.
  • the present disclosure is also based, in part, on the use of a hybrid promoter that allows the expression of a gRNA in live cells and transcription of the gRNA in fixed cells without effecting the editing efficiency of the gRNA.
  • the present disclosure is further based, in part, on using decrosslinking to allow the use of standard phenotyping protocols, e.g., use of antibodies, oligonucleotides and dyes for phenotyping, to be performed prior to performing in situ sequencing to visualize target nucleic acids.
  • decrosslinking allows for the use of additional types of fixatives, e.g., including aldehyde-based fixatives, prior to performing in vitro transcription.
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, z.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.
  • amplification process refers generally to any process where a portion of a nucleic acid is copied or replicated into at least one additional nucleic acid molecule.
  • antibody herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments so long as they exhibit the desired antigen-binding activity.
  • antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments comprise but are not limited to Fv, Fab, Fab’, Fab’- SH, F(ab’)2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv) and multispecific antibodies formed from antibody fragments.
  • Coupled can refer to the connecting or uniting of two or more components by an interaction, bond, link, force or tie in order to keep two or more components together.
  • the term “coupled” encompasses either direct or indirect binding where, for example, a first component is directly bound to a second component, or one or more intermediate molecules are disposed between the first component and the second component.
  • Exemplary bonds comprise covalent bonds, ionic bonds, van der Waals interactions and other bonds identifiable by a skilled person.
  • detect or “detection,” as used herein, indicate the determination of the existence and/or presence of a target, e.g., a nucleic acid target, in a limited portion of space, including but not limited to a sample.
  • a target e.g., a nucleic acid target
  • detection can comprise determination of chemical and/or biological properties of the target, including but not limited to ability to interact, and in particular bind, other compounds, ability to activate another compound and additional properties identifiable by a skilled person upon reading of the present disclosure.
  • the detection can be quantitative or qualitative.
  • a detection is “quantitative” when it refers, relates to, or involves the measurement of quantity or amount of the target or signal (also referred as quantitation), which comprises but is not limited to any analysis designed to determine the amounts or proportions of the target or signal.
  • a detection is “qualitative” when it refers, relates to, or involves identification of a quality or kind of the target or signal in terms of relative abundance to another target or signal, which is not quantified.
  • editing efficiency refers to the total number of sequence reads with insertions or deletions of nucleotides into a target region of interest over the total number of sequence reads following cleavage by an RNA-guided nuclease.
  • guide RNA refers to a nucleic acid that promotes the specific targeting or homing of an RNA- guided nuclease to a target nucleic acid.
  • hybridization refers to the process in which two singlestranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide.
  • hybrid promoter refers to a promoter sequence, e.g., a promoter nucleotide sequence, that comprises nucleotide sequences (or portions thereof) derived from at least two different promoters.
  • a “hybrid promoter” of the present disclosure comprises a nucleotide sequence (or a portion thereof) derived from at least one promoter for expressing a nucleic acid in live cells and comprises a nucleotide sequence (or a portion thereof) derived from at least one promoter for expressing the nucleic acid in fixed cells.
  • a hybrid promoter of the present disclosure includes a nucleotide sequence (or a portion thereof) derived from at least one mammalian promoter (e.g., for expressing a nucleic acid in live cells) and comprises a nucleotide sequence (or a portion thereof) derived from at least one bacteriophage promoter (e.g., for expressing the nucleic acid in fixed cells).
  • a hybrid promoter is a promoter that includes a nucleotide sequence (or a portion thereof) derived from a second promoter that is incorporated into the nucleotide sequence (or a portion thereof) derived from a first promoter.
  • microscopy refers to microscopy.
  • microscopy comprises immunofluorescence microscopy.
  • microscopy comprises optical microscopy.
  • the term “individual” or “subject” refers to a vertebrate or an invertebrate, such as a human or non-human animal, for example, a mammal. Mammals comprise, but are not limited to, humans, non-human primates, farm animals, sport animals, rodents and pets. Non-limiting examples of non-human animal subjects comprise rodents such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cattle, horses, apes and monkeys. In certain embodiments, the individual or subject is a human.
  • z z vitro refers to an artificial environment and to processes or reactions that occur within an artificial environment.
  • In vitro environments exemplified, but are not limited to, test tubes and cell cultures.
  • a “label” refers to an agent that allows for direct or indirect detection. Labels comprise, but are not limited to, fluorescent labels, chromogenic labels, electron dense labels, chemiluminescent labels and radioactive labels. Non-limiting examples of labels comprise green fluorescent protein (“GFP”), mCherry, dtTomato, or other fluorescent proteins known in the art e.g., Shaner et al., A Guide to Choosing Fluorescent Proteins, Nature Methods 2(12):905-909 (2005) incorporated by reference herein, 32 P, 14 C, 125 1, 3 H and 131 I, fluorogens (such as Rare Earth Chelate or lucifer yellow and its derivatives), Rhodamine (rhodamine) and its derivatives, dansyl, umbelliferone, luciferase (such as firefly luciferase and bacterial fluorescence plain enzyme) (U.S.
  • GFP green fluorescent protein
  • mCherry mCherry
  • Patent number 4,737,456 fluorescein, 2,3-dihydros phthalazine diketone, as well as enzymes producing detectable signals, e.g., horseradish peroxidase (HRP), alkaline phosphorus sour enzyme, beta galactosidase, glucoamylase, lysozyme, carbohydrate oxidase (such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase (G6PD)) and heterocyclic oxidases (such as uricase and xanthine oxidase).
  • HRP horseradish peroxidase
  • alkaline phosphorus sour enzyme beta galactosidase
  • glucoamylase lysozyme
  • carbohydrate oxidase such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase (G6
  • ligation refers to the formation of a covalent bond or linkage between two or more molecules, e.g., between the termini of two or more nucleic acid molecules.
  • ligation process refers generally to a process for covalently linking two or more molecules together by an enzyme.
  • two or more nucleic acid molecules can be covalently linked together by a ligation process using a ligase.
  • the term “monoclonal antibody,” as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations which typically comprise different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the presently disclosed subject matter may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
  • nucleic acid or “polynucleotide” comprises any compound and/or substance that comprises a polymer of nucleotides.
  • Each nucleotide is composed of a base, specifically a purine- or pyrimidine base (z.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (z.e., deoxyribose or ribose), and a phosphate group.
  • cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U) a sugar
  • z.e., deoxyribose or ribose a phosphate group.
  • the nucleic acid molecule is described by the sequence of bases, whereby said bases represent the primary structure (linear structure) of a nucleic acid molecule.
  • nucleic acid encompasses deoxyribonucleic acid (DNA) including, e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), e.g., messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • the sequence of bases is typically represented from 5’ to 3’.
  • nucleic acid encompasses deoxyribonucleic acid (DNA) including, e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), e.g., messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules.
  • the nucleic acid molecule can be linear or circular.
  • nucleic acid comprises both, sense and antisense strands, as well as single stranded and double strande
  • operative connection with regard to regulatory sequences of a nucleic acid, e.g., a gene, indicate an arrangement of elements in a combination enabling production of an appropriate effect.
  • an operative connection indicates a configuration of the nucleic acids, e.g., genes, with respect to the regulatory sequence allowing the regulatory sequences to directly or indirectly increase or decrease transcription or translation of the nucleic acids, e.g., genes.
  • regulatory sequences directly increasing transcription of the operatively linked nucleic acid comprise promoters typically located on a same strand and upstream on a DNA sequence (towards the 5’ region of the sense strand), adjacent to the transcription start site of the nucleic acids, e.g., genes, whose transcription they initiate.
  • regulatory sequences directly increasing transcription of the operatively linked nucleic acid, e.g., gene comprise enhancers that can be located more distally from the transcription start site compared to promoters, and either upstream or downstream from the regulated nucleic acids, e.g., genes, as understood by those skilled in the art.
  • Enhancers are typically short (50-1500 bp) regions of DNA that can be bound by transcriptional activators to increase transcription of a particular nucleic acid, e.g., gene. Typically, enhancers can be located up to 1 Mbp away from the nucleic acid, e.g., gene, upstream or downstream from the start site.
  • percentage of sequence identity or “percentage of identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window can comprise additions or deletions (gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
  • determination of percent identity between any two sequences can be accomplished using certain well-known mathematical algorithms. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller, the local homology algorithm of Smith et al.; the homology alignment algorithm of Needleman and Wunsch; the search-for-similarity-method of Pearson and Lipman; the algorithm of Karlin and Altschul, modified as in Karlin and Altschul.
  • Computer implementations of suitable mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations comprise, but are not limited to: CLUSTAL, ALIGN, GAP, BESTFIT, BLAST, FASTA, among others identifiable by skilled persons. Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Molecular Biol., 215(3): 403-410 (1990); Beigert et al., Proc. Natl. Acad. Sci.
  • the term “plurality” refers to a number larger than one.
  • the term “plurality of cells” refers to a number of cells larger than one.
  • a plurality of proteins comprises at least two cells.
  • the term “plurality of nucleic acids” refers to a number of nucleic acids larger than one.
  • a plurality of nucleic acids comprises at least two nucleic acids.
  • the term “plurality of guide RNAs” refers to a number of guide RNAs larger than one.
  • a plurality of guide RNAs comprises at least two guide RNAs.
  • the term “reverse-transcription process” refers to a process of generating a complementary strand of DNA using an enzyme called a reverse transcriptase.
  • sample refers to any sample containing one or more individual cells.
  • sample refers to a sample of biological material obtained from a subject, e.g., a tissue biopsy or a tissue sample.
  • the sample can be obtained from a tissue, e.g., a tissue sample.
  • Non-limiting examples of tissues comprise eye, muscle, skin, tendon, vein, artery, blood, heart, spleen, lymph node, bone, bone marrow, lung, bronchi, trachea, gut, small intestine, large intestine, colon, rectum, salivary gland, tongue, gallbladder, appendix, liver, pancreas, brain, stomach, skin, kidney, ureter, bladder, urethra, gonad, testicle, ovary, uterus, fallopian tube, thymus, pituitary, thyroid, adrenal or parathyroid tissue.
  • the samples are obtained from a subject.
  • the subject can be a human, non-human primate, e.g., an ape or a monkey, a farm animal, a mouse, a rat, a hamster, a guinea pig, a rabbit, a dog, cat, a sheep, a pig, a goat, a cow or a horse.
  • the subject is a human.
  • the sample can be obtained from preserved tissue, e.g., fixed tissue, from frozen tissue or from fresh tissue, e.g., tissue samples.
  • a sample that can be analyzed using the methods of the present disclosure comprise at least two or more cells.
  • a sample can comprise about 10 or more cells, about 100 or more cells, about 1,000 or more cells, about 5,000 or more cells, about 10,000 or more cells, about 20,000 or more cells, about 30,000 or more cells, about 40,000 or more cells, about 50,000 or more cells, about 100,000 or more cells, about 150,000 or more cells, about 200,000 or more cells, about 300,000 or more cells, about 400,000 or more cells or 500,000 or more cells.
  • the cells of a sample are obtained from (e.g., isolated from) a tissue.
  • sequence identity or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to the nucleotide bases or amino acid residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • sequence identity or similarity in reference to proteins, it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted with a functionally equivalent residue of the amino acid residues with similar physiochemical properties and therefore do not change the functional properties of the molecule.
  • telomere binding refers to the preferential binding to a target molecule, e.g., a protein or nucleic acid, relative to other molecules, e.g., proteins or nucleic acids, in a sample.
  • treatment refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology.
  • Desirable effects of treatment comprise, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • the decrease can be at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% decrease in severity of complications, signs or symptoms or in likelihood of progression to another grade. “Treatment” can also refer to inhibiting proliferation of a cancer or progression to a higher grade by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%.
  • gRNAs identified by methods of the present disclosure can used to delay development of a disease or to slow the progression of a disease.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term comprises the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
  • the present disclosure provides nucleic acid constructs for use in the methods of the present disclosure.
  • the present disclosure further provides compositions (e.g., nucleic acid compositions) that comprise one or more nucleic acid constructs for use in the methods of the present disclosure.
  • a nucleic acid construct of the present disclosure comprises one or more target nucleic acids and/or two or more promoters. In certain embodiments, a nucleic acid construct of the present disclosure comprises two or more promoters. In certain embodiments, a nucleic acid construct of the present disclosure comprises one or more target nucleic acids. In certain embodiments, a nucleic acid construct of the present disclosure comprises one or more target nucleic acids and two or more promoters.
  • a nucleic acid construct of the present disclosure comprises a target nucleic acid.
  • a nucleic acid construct of the present disclosure comprises a target nucleic acid that is to be detected and/or visualized by a method disclosed herein.
  • the target nucleic acid can be about 2 to about 10,000 nucleotides, e.g., about 2 to about 1,000 nucleotides, in length.
  • the target nucleic acid can be about 10 to about 10,000 nucleotides, about 100 to about 10,000 nucleotides, about 200 to about 10,000 nucleotides, about 300 to about 10,000 nucleotides, about 400 to about 10,000 nucleotides, about 500 to about 10,000 nucleotides, about 600 to about 10,000 nucleotides, about 700 to about 10,000 nucleotides, about 800 to about 10,000 nucleotides, about 900 to about 10,000 nucleotides, about 1,000 to about 10,000 nucleotides, about 2,000 to about 10,000 nucleotides, about 3,000 to about 10,000 nucleotides, about 4,000 to about 10,000 nucleotides, about 5,000 to about 10,000 nucleotides, about 6,000 to about 10,000 nucleotides, about 7,000 to about 10,000 nucleotides, about 8,000 to about 10,000 nucleotides, about 9,000 to about 10,000 nucleotides, about 10 to about 9,000 nucleotides, about 10 to about 8,000 nucleotides,
  • the target nucleic acid can be about 2 to about 900 nucleotides, about 2 to about 800 nucleotides, about 2 to about 700 nucleotides, about 2 to about 600 nucleotides, about 2 to about 500 nucleotides, about 2 to about 400 nucleotides, about 2 to about 300 nucleotides, about 2 to about 200 nucleotides, about 2 to about 150 nucleotides, about 2 to about 100 nucleotides, about 2 to about 90 nucleotides, about 2 to about 80 nucleotides, about 2 to about 70 nucleotides, about 2 to about 60 nucleotides, about 2 to about 90 nucleotides, about 50 to about 900 nucleotides, about 50 to about 800 nucleotides, about 50 to about 700 nucleotides, about 50 to about 600 nucleotides, about 50 to about 500 nucleotides, about 50 to about 400 nucleotides, about 50 to about 300 nucleot
  • the target nucleic acid visualized by the methods of the present disclosure can comprise about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 60 or more, about 80 or more, about 100 or more, about 150 or more, about 200 or more, about 300 or more, about 400 or more, about 500 or more, about 1,000 or more, about 2,000 or more, about 3,000 or more, about 4,000 or more, about 5,000 or more, about 6,000 or more, about 7,000 or more, about 8,000 or more, about 9,000 or more or about 10,000 or more nucleotides in length.
  • the target nucleic acid can be about 1 to about 1,000 nucleotides in length.
  • the target nucleic acid can be a nucleic acid that when expressed affects the expression of a gene and/or affects the levels of a messenger RNA (mRNA).
  • the target nucleic acid can be a nucleic acid that when expressed results in the knock down or knock out of a gene.
  • the target nucleic acid can be a nucleic acid that when expressed results in gene activation.
  • the target nucleic acid can be a nucleic acid that when expressed results in the insertion of a polynucleotide into a genomic sequence.
  • the target nucleic acid can be a nucleic acid that when expressed results in the deletion of a genomic sequence.
  • the target nucleic acid can be a nucleic acid that when expressed affects the expression, cellular localization and/or post- translational modification of a protein.
  • the target nucleic acid is a non-coding RNA.
  • the target nucleic acid can be a guide RNA (gRNA), a microRNA (miRNA), a small nucleolar RNA (snoRNA), a small interfering RNA (siRNA), a piwi-interacting RNA (piRNA), an aptamer, a ribozyme, an endogenous siRNA (endo-siRNA) or a short hairpin RNA (shRNA), a generic barcode indicating the identity of a larger RNA or DNA molecule that the barcode is a component of, e.g., a sequence of, an mRNA encoding a protein of interest.
  • the target nucleic acid can be a barcode, e.g., a generic barcode.
  • a generic barcode is a barcode comprising random nucleotides or a barcode comprising a designed series of nucleotides.
  • a target nucleic acid that is detected by methods of the present disclosure can be a nucleic acid that comprises a nucleotide sequence that is at least partially complementary to or at least partially identical a known nucleotide sequence.
  • a target nucleic acid can comprise a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary to a known sequence, e.g., a genomic sequence.
  • a target nucleic acid can comprise a nucleotide sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to a known sequence, e.g., a genomic sequence.
  • the genomic sequence can be an intron or an exon.
  • the genomic sequence can be a regulatory sequence of a gene, e.g., a promoter and/or an enhancer.
  • the target nucleic acid encodes a gRNA.
  • a gRNA that is encoded by a nucleic acid construct of the present disclosure has a length from about 20 to about 200 nucleotides, e.g., from about 20 to about 190, from about 20 to about 180, from about 20 to about 170, from about 20 to about 160, from about 20 to about 150, from about 20 to about 140, from about 20 to about 130, from about 20 to about 120, from about 20 to about 110, from about 20 to about 100, from about 30 to about 200, from about 40 to about 200, from about 50 to about 200, from about 60 to about 200, from about 70 to about 200, from about 80 to about 200, from about 90 to about 200, from about 50 to about 150, from about 80 to about 120 or from about 90 to about 100 nucleotides.
  • a gRNA of the present disclosure has a length from about 80 to about 120 nucleotides. In certain embodiments, a gRNA of the present disclosure is about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195 or about 200 or more nucleotides in length.
  • the gRNA comprises a targeting domain that is complementary to, e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary to, a genomic nucleotide sequence.
  • the targeting domain is from about 15 to about 25 nucleotides in length. In certain embodiments, the targeting domain is 18 nucleotides in length.
  • the targeting domain is 19 nucleotides in length. In certain embodiments, the targeting domain is 20 nucleotides in length. In certain embodiments, the targeting domain is 21 nucleotides in length. In certain embodiments, the targeting domain is 22 nucleotides in length. In certain embodiments, the targeting domain is 23 nucleotides in length. In certain embodiments, the targeting domain is 24 nucleotides in length. In certain embodiments, the targeting domain is 25 nucleotides in length.
  • a gRNA of the present disclosure can have a scaffold as disclosed in Dang et al., Genome Biology 16:280 (2015), the contents of which are incorporated by reference herein in their entirety.
  • the duplex region of the gRNA scaffold can be extended by at least 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 6 base pairs, 7 base pairs, 8 base pairs, 9 base pairs or 10 base pairs (see Figure 1 of Dang et al. (2015)).
  • the duplex region of the gRNA scaffold can be extended by at least 5 base pairs, e.g., to improve editing efficiency of the gRNA (see Figure 7 of Dang et al. (2015)).
  • the gRNA of the present disclosure can further include one or more mutations in the duplex (e.g. , in the lower stem of the duplex).
  • the gRNA scaffold of the present disclosure can include a mutation in the continuous sequence of Ts present in the duplex (e.g., present in the lower stem of the duplex), as shown in Figure 7 of Dang et al. (2015), to improve editing efficiency of the gRNA.
  • position 4 of the continuous sequence of Ts in the lower stem of the duplex is mutated to a C or G).
  • a gRNA of the present disclosure can have an editing efficiency of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%
  • the gRNA has an editing efficiency of about 60% or greater (e.g., when expressed using a nucleic acid construct of the present disclosure (e.g., under control of a hybrid promoter disclosed herein)). In certain embodiments, the gRNA has an editing efficiency of about 65% or greater (e.g., under control of a hybrid promoter disclosed herein)). In certain embodiments, the gRNA has an editing efficiency of about 70% or greater (e.g., when expressed using a nucleic acid construct of the present disclosure (e.g., under control of a hybrid promoter disclosed herein)).
  • the gRNA has an editing efficiency of about 75% or greater (e.g., when expressed using a nucleic acid construct of the present disclosure (e.g., under control of a hybrid promoter disclosed herein)). In certain embodiments, the gRNA has an editing efficiency of about 80% or greater. In certain embodiments, the gRNA has an editing efficiency of about 85% or greater (e.g., when expressed using a nucleic acid construct of the present disclosure (e.g., under control of a hybrid promoter disclosed herein)).
  • the gRNA has an editing efficiency of about 90% or greater (e.g., when expressed using a nucleic acid construct of the present disclosure (e.g., under control of a hybrid promoter disclosed herein)). In certain embodiments, the gRNA has an editing efficiency of about 95% or greater (e.g., when expressed using a nucleic acid construct of the present disclosure (e.g., under control of a hybrid promoter disclosed herein)).
  • the methods of the present disclosure can be used to identify gRNAs that have an editing efficiency of about 5% or greater, about 10% or greater, about
  • a nucleic acid construct of the present disclosure comprises at least two promoters.
  • a nucleic acid construct of the present disclosure comprises at least two promoters located upstream of the target nucleic acid, e.g., as shown in FIG. 3B.
  • a nucleic acid construct of the present disclosure comprises a first promoter and a second promoter located upstream of the target nucleic acid, e.g., operably linked to the target nucleic acid.
  • a nucleic acid construct of the present disclosure comprises at least two promoters located immediately 5’ to the nucleotide sequence of the target nucleic acid.
  • the first promoter and the second promoter in a nucleic acid construct of the present disclosure are different.
  • the first promoter and the second promoter are recognized by different polymerases, e.g., different RNA polymerases.
  • one of the promoters for use in the present disclosure is configured to express the target nucleic acid in live cells.
  • the first promoter is a promoter active in live cells.
  • live cells comprise cells that are actively dividing, have intact membranes and/or are metabolically active.
  • the first promoter is a promoter that is inactive in fixed cells, e.g., cells that have been contacted with a fixative.
  • the first promoter is a regulated promoter (e.g., inducible promoter).
  • the first promoter is a constitutive promoter.
  • the first promoter is a viral promoter.
  • the first promoter is a mammalian promoter. In certain embodiments, the first promoter is a non-viral promoter.
  • Non-limiting examples of promoters for expressing the target nucleic acid in a live cell comprise U6, U3, U2, U5, Hl, 75J, EF-la, CMV, tRNA promoters, pGK, SV40, CAG, TRE, 7SK and VAI.
  • the first promoter can be a promoter recognized by an RNA polymerase. In certain embodiments, the first promoter is recognized by RNA polymerase II (e.g. , a CMV promoter), e.g. , a Pol II promoter.
  • the first promoter is recognized by RNA polymerase III (e.g. , a U6 promoter), e.g., a Pol III promoter.
  • RNA polymerase III e.g. , a U6 promoter
  • the promoter for expressing the target nucleic acid in live cells is a U6 promoter.
  • an additional promoter for use in the present disclosure is configured to express the target nucleic acid in fixed cells (e.g., a second promoter).
  • fixed cells are cells that have been contacted with a fixative.
  • the second promoter is a promoter that is active in fixed cells.
  • the second promoter is a promoter inactive in live cells.
  • the second promoter is a regulated promoter (e.g., inducible promoter).
  • the second promoter is a constitutive promoter.
  • the second promoter is a viral promoter.
  • the second promoter is a non-viral promoter.
  • the second promoter can be a promoter for a phage RNA polymerase.
  • phage RNA polymerases comprise a bacteriophage T3 RNA polymerase, a bacteriophage T7 RNA polymerase, a bacteriophage SP6 RNA polymerase or a combination thereof.
  • the second promoter can be a T3 promoter, a T7 promoter and/or a Sp6 promoter.
  • the promoter for expressing the target nucleic acid in fixed cells e.g., a second promoter
  • the promoter for expressing the target nucleic acid in fixed cells is a T7 promoter.
  • the first promoter is a promoter active in live cells and the second promoter is a promoter active in fixed cells.
  • the first promoter is a mammalian promoter and the second promoter is a bacteriophage promoter.
  • the promoter for expressing the target nucleic acid in fixed cells is located upstream to, downstream to or integrated into the promoter being used to express the target nucleic acid in live cells (e.g., the first promoter).
  • the promoter for expressing the target nucleic acid in fixed cells is located upstream to the promoter being used to express the target nucleic acid in live cells (e.g., the first promoter).
  • the promoter for expressing the target nucleic acid in fixed cells is located downstream to the promoter being used to express the target nucleic acid in live cells (e.g., the first promoter). In certain embodiments, the promoter for expressing the target nucleic acid in fixed cells (e.g., the second promoter) is integrated into the promoter being used to express the target nucleic acid in live cells (e.g., the first promoter).
  • the promoter for expressing the target nucleic acid in fixed cells is integrated into the promoter being used to express the target nucleic acid in live cells (e.g., the first promoter) to generate a hybrid promoter.
  • live cells e.g., the first promoter
  • nucleic acids that comprise the nucleotide sequences of the first promoter and the second promoter are provided in FIG. 3B.
  • the nucleotide sequence of the second promoter is integrated into the nucleotide sequence of the first promoter, e.g., to generate a hybrid promoter. In certain embodiments, the nucleotide sequence of the second promoter is integrated into the nucleotide sequence of the first promoter at a position that does not affect the function of the first promoter, e.g., the ability of the first promoter to initiate expression of the target nucleic acid (e.g., in live cells).
  • the nucleotide sequence of the second promoter is integrated into the nucleotide sequence of the first promoter at a position that does not affect the function of the second promoter, e.g., the ability of the second promoter to initiate expression of the target nucleic acid (e.g., in fixed cells).
  • incorporation of the nucleotide sequence of the second promoter (e.g., T7 promoter) into the nucleotide sequence of the first promoter did not reduce the editing efficiency of the expressed gRNA as compared to the editing efficiency of the gRNA when expressed using the wild-type U6 promoter.
  • a second promoter e.g., T7 promoter
  • the sequence of the first promoter e.g., U6 promoter
  • the U6 pub promoter referred to as the U6 pub promoter herein
  • the use of a nucleic acid construct comprising a hybrid promoter described herein does not reduce the editing efficiency of the gRNA under control of the hybrid promoter by more than about 30% (e.g., by more than about 25%, by more than about 20%, by more than about 15%, by more than about 10% or by more than about 5%) compared to the editing efficiency of the gRNA under control of the wild-type U6 promoter. In certain embodiments, the use of a nucleic acid construct comprising a hybrid promoter described herein does not reduce the editing efficiency of the gRNA under control of the hybrid promoter by more than about 20% compared to the editing efficiency of the gRNA under control of the wild-type U6 promoter.
  • the use of a nucleic acid construct comprising a hybrid promoter described herein does not reduce the editing efficiency of the gRNA under control of the hybrid promoter by more than about 10% compared to the editing efficiency of the gRNA under control of the wild-type U6 promoter. In certain embodiments, the use of a nucleic acid construct comprising a hybrid promoter described herein does not reduce the editing efficiency of the gRNA under control of the hybrid promoter by more than about 5% compared to the editing efficiency of the gRNA under control of the wild-type U6 promoter.
  • the nucleotide sequence of the second promoter can be inserted within the first 10 nucleotides located at the 5’ end of the nucleotide sequence of the first promoter, e.g., within the first 20 nucleotides, within the first 30 nucleotides, within the first 40 nucleotides, within the first 50 nucleotides, within the first 60 nucleotides, within the first 70 nucleotides, within the first 80 nucleotides, within the first 90 nucleotides, within the first 100 nucleotides, within the first 110 nucleotides, within the first 120 nucleotides, within the first 130 nucleotides, within the first 140 nucleotides, within the first 150 nucleotides, within the first 160 nucleotides, within the first 170 nucleotides, within the first 180 nucleotides, within the first 190 nucleotides, within the first 200 nucleotides, within the first 210 nucle
  • the nucleotide sequence of the second promoter can be inserted within the first 100 nucleotides located at the 5’ end of the nucleotide sequence of the first promoter. In certain embodiments, the nucleotide sequence of the second promoter can be inserted within the first 150 nucleotides located at the 5’ end of the nucleotide sequence of the first promoter. In certain embodiments, the nucleotide sequence of the second promoter can be inserted within the first 200 nucleotides located at the 5’ end of the nucleotide sequence of the first promoter.
  • the second promoter can be inserted within the last 10 nucleotides located at the 3 ’ end of the nucleotide sequence of the first promoter, e.g. , within the last 20 nucleotides, within the last 30 nucleotides, within the last 40 nucleotides, within the last 50 nucleotides, within the last 60 nucleotides, within the last 70 nucleotides, within the last 80 nucleotides, within the last 90 nucleotides, within the last 100 nucleotides, within the last 110 nucleotides, within the last 120 nucleotides, within the last 130 nucleotides, within the last 140 nucleotides, within the last 150 nucleotides, within the last 160 nucleotides, within the last 170 nucleotides, within the last 180 nucleotides, within the last 190 nucleotides, within the last 200 nucleotides, within the last 210 nucleotides, within the
  • the second promoter can be inserted within the last 50 nucleotides located at the 3’ end of the nucleotide sequence of the first promoter. In certain embodiments, the nucleotide sequence of the second promoter can be inserted within the last 100 nucleotides located at the 3 ’ end of the nucleotide sequence of the first promoter.
  • the nucleotide sequence of the second promoter can be inserted between nucleotides located about 40 to about 200 nucleotides downstream from the 5’ end of the nucleotide sequence of the first promoter, e.g., to generate a hybrid promoter. In certain embodiments, the nucleotide sequence of the second promoter can be inserted between nucleotides located about 50 to about 200 nucleotides downstream from the 5’ end of the nucleotide sequence of the first promoter, e.g., to generate a hybrid promoter.
  • the nucleotide sequence of the second promoter can be inserted between nucleotides located about 40 to about 190 nucleotides downstream from the 5’ end of the nucleotide sequence of the first promoter, e.g., to generate a hybrid promoter. In certain embodiments, the nucleotide sequence of the second promoter can be inserted between nucleotides located about 100 to about 200 nucleotides downstream from the 5’ end of the nucleotide sequence of the first promoter. In certain embodiments, the nucleotide sequence of the second promoter can be inserted between nucleotides located about 150 to about 200 nucleotides downstream from the 5’ end of the nucleotide sequence of the first promoter.
  • the nucleotide sequence of the second promoter can be inserted between a SPH element and a TATA box of the first promoter, e.g., to generate a hybrid promoter.
  • the nucleotide sequence of the second promoter can be inserted between an octamer (OCT) element and a TATA box of the first promoter, e.g., to generate a hybrid promoter.
  • the second promoter can be inserted between a SPH element and a PSE element of the first promoter, e.g., to generate a hybrid promoter.
  • the nucleotide sequence of the second promoter can be inserted between an OCT element and a PSE element of the first promoter, e.g., to generate a hybrid promoter. In certain embodiments, the nucleotide sequence of the second promoter can be inserted between the TATA box and the 3’ end of the first promoter, e.g., to generate a hybrid promoter.
  • a nucleic acid construct of the present disclosure can comprise a T7 promoter as the second promoter.
  • the T7 promoter has the nucleotide sequence TAATACGACTCACTATAG (SEQ ID NO: 1).
  • the T7 promoter has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 1.
  • the T7 promoter has at least about 85% sequence identity to the nucleotide sequence of SEQ ID NO: 1. In certain embodiments, the T7 promoter has at least about 90% sequence identity to the nucleotide sequence of SEQ ID NO: 1. In certain embodiments, the T7 promoter has at least about 95% sequence identity to the nucleotide sequence of SEQ ID NO: 1. In certain embodiments, the T7 promoter has at least about 96% sequence identity to the nucleotide sequence of SEQ ID NO: 1. In certain embodiments, the T7 promoter has at least about 97% sequence identity to the nucleotide sequence of SEQ ID NO: 1.
  • the T7 promoter has at least about 98% sequence identity to the nucleotide sequence of SEQ ID NO: 1. In certain embodiments, the T7 promoter has at least about 99% sequence identity to the nucleotide sequence of SEQ ID NO: 1.
  • a nucleic acid construct of the present disclosure can comprise a Sp6 promoter as the second promoter. In certain embodiments, the Sp6 promoter has the nucleotide sequence ATTTAGGTGACACTATAG (SEQ ID NO: 2).
  • the Sp6 promoter has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 2. In certain embodiments, the Sp6 promoter has at least about 85% sequence identity to the nucleotide sequence of SEQ ID NO: 2. In certain embodiments, the Sp6 promoter has at least about 90% sequence identity to the nucleotide sequence of SEQ ID NO: 2.
  • the Sp6 promoter has at least about 95% sequence identity to the nucleotide sequence of SEQ ID NO: 2. In certain embodiments, the Sp6 promoter has at least about 96% sequence identity to the nucleotide sequence of SEQ ID NO: 2. In certain embodiments, the Sp6 promoter has at least about 97% sequence identity to the nucleotide sequence of SEQ ID NO: 2. In certain embodiments, the Sp6 promoter has at least about 98% sequence identity to the nucleotide sequence of SEQ ID NO: 2. In certain embodiments, the Sp6 promoter has at least about 99% sequence identity to the nucleotide sequence of SEQ ID NO: 2.
  • a nucleic acid construct of the present disclosure can comprise a T3 promoter as the second promoter.
  • the T3 promoter has the nucleotide sequence AATTAACCCTCACTAAAG (SEQ ID NO: 3).
  • the T3 promoter has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 3.
  • the T3 promoter has at least about 85% sequence identity to the nucleotide sequence of SEQ ID NO: 3. In certain embodiments, the T3 promoter has at least about 90% sequence identity to the nucleotide sequence of SEQ ID NO: 3. In certain embodiments, the T3 promoter has at least about 95% sequence identity to the nucleotide sequence of SEQ ID NO: 3. In certain embodiments, the T3 promoter has at least about 96% sequence identity to the nucleotide sequence of SEQ ID NO: 3. In certain embodiments, the T3 promoter has at least about 97% sequence identity to the nucleotide sequence of SEQ ID NO: 3.
  • the T3 promoter has at least about 98% sequence identity to the nucleotide sequence of SEQ ID NO: 3. In certain embodiments, the T3 promoter has at least about 99% sequence identity to the nucleotide sequence of SEQ ID NO: 3.
  • a nucleic acid construct of the present disclosure can comprise a U6 promoter as the first promoter.
  • the U6 promoter is derived from mouse or human.
  • the U6 promoter is the human U6 promoter.
  • the U6 promoter comprises the following nucleotide sequence
  • the U6 promoter is the U6 promoter shown in FIG. 3B and comprises the nucleotide sequence of SEQ ID NO: 4.
  • the U6 promoter is the mini U6 promoter and has the following nucleotide sequence GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATAG CTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAA GGAC (SEQ ID NO: 5).
  • the U6 promoter has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 4 or 5.
  • the U6 promoter has at least about 85% sequence identity to the nucleotide sequence of SEQ ID NO: 4 or 5. In certain embodiments, the U6 promoter has at least about 90% sequence identity to the nucleotide sequence of SEQ ID NO: 4 or 5. In certain embodiments, the U6 promoter has at least about 95% sequence identity to the nucleotide sequence of SEQ ID NO: 4 or 5. In certain embodiments, the U6 promoter has at least about 96% sequence identity to the nucleotide sequence of SEQ ID NO: 4 or 5. In certain embodiments, the U6 promoter has at least about 97% sequence identity to the nucleotide sequence of SEQ ID NO: 4 or 5.
  • the U6 promoter has at least about 98% sequence identity to the nucleotide sequence of SEQ ID NO: 4 or 5. In certain embodiments, the U6 promoter has at least about 99% sequence identity to the nucleotide sequence of SEQ ID NO: 4 or 5.
  • a nucleic acid construct of the present disclosure can comprise a hybrid promoter comprising the sequence of a T7 promoter inserted into the sequence of a U6 promoter.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter, can comprise a nucleotide sequence shown in FIG. 3B and Table 2.
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to a nucleotide sequence shown in FIG. 3B and Table 2.
  • the nucleic acid construct comprising the at least two promoters e.g.
  • a hybrid promoter can have a nucleotide sequence that has at least about 85% sequence identity to a nucleotide sequence shown in FIG. 3B and Table 2.
  • the nucleic acid construct comprising the at least two promoters e.g, a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 90% sequence identity to a nucleotide sequence shown in FIG. 3B and Table 2.
  • the nucleic acid construct comprising the at least two promoters, e.g, a hybrid promoter can have a nucleotide sequence that has at least about 95% sequence identity to the nucleotide sequence shown in FIG. 3B and Table 2.
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 96% sequence identity to a nucleotide sequence shown in FIG. 3B and Table 2.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 98% sequence identity to a nucleotide sequence shown in FIG. 3B and Table 2.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters, e.g., a hybrid promoter can have a nucleotide sequence shown in FIG. 3B and Table 2.
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of
  • the nucleic acid construct comprising at least two promoters is the U6 pub shown in FIG. 3B and/or Table 2 and comprises the nucleotide sequence of SEQ ID NO: 6.
  • the sequence of T7 promoter is located 3’ to the nucleotide sequence of the U6 promoter and is not incorporated into the nucleotide sequence of U6 promoter, as shown in FIG. 3B.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter, does not comprise or consist of the nucleotide sequence of SEQ ID NO: 6.
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of
  • nucleic acid construct comprising the at least two promoters, e.g.
  • a hybrid promoter can have a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTT AGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAA ATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTTAATACG ACTCACTATAGGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTC TTGGCTTTATATATCTTGTGGAAAGGAC (SEQ ID NO: 8).
  • the nucleic acid construct comprising the at least two promoters is the U6 vl promoter shown in FIG. 3B and/or Table 2 and comprises the nucleotide sequence of SEQ ID NO: 7 or 8.
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of
  • the nucleic acid construct comprising the at least two promoters is the U6 v2 promoter shown in FIG. 3B and/or Table 2 and comprises the nucleotide sequence of SEQ ID NO: 9.
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of
  • the nucleic acid construct comprising the at least two promoters is the U6 v3 promoter shown in FIG. 3B and/or Table 2 and comprises the nucleotide sequence of SEQ ID NO: 10.
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of
  • the nucleic acid construct comprising the at least two promoters is the U6 v4 promoter shown in FIG. 3B and comprises the nucleotide sequence of SEQ ID NO: 11.
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of
  • the nucleic acid construct comprising the at least two promoters is the U6 v5 promoter shown in FIG. 3B and/or Table 2 and comprises the nucleotide sequence of SEQ ID NO: 12.
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of
  • the nucleic acid construct comprising the at least two promoters is the U6 v6 promoter shown in FIG. 3B and/or Table 2 and comprises the nucleotide sequence of SEQ ID NO: 13.
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of
  • the nucleic acid construct comprising the at least two promoters is the U6 v7 promoter shown in FIG. 3B and/or Table 2 and comprises the nucleotide sequence of SEQ ID NO: 14.
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of
  • the nucleic acid construct comprising the at least two promoters is the U6 v8 promoter shown in FIG. 3B and/or Table 2 and comprises the nucleotide sequence of SEQ ID NO: 15.
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of
  • the nucleic acid construct comprising the at least two promoters is the U6 v9 promoter shown in FIG. 3B and/or Table 2 and comprises the nucleotide sequence of SEQ ID NO: 16.
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of
  • the nucleic acid construct comprising the at least two promoters is the U6 v10 promoter shown in FIG. 3B and/or Table 2 and comprises the nucleotide sequence of SEQ ID NO: 17.
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of
  • the nucleic acid construct comprising the at least two promoters is the U6 v11 promoter shown in FIG. 3B and/or Table 2 and comprises the nucleotide sequence of SEQ ID NO: 18.
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 85% sequence identity to the nucleotide sequence of SEQ ID NOs: 6-18.
  • the nucleic acid construct comprising the at least two promoters e.g. , a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 85% sequence identity to the nucleotide sequence of SEQ ID NO: 6.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters e.g.
  • a hybrid promoter can have a nucleotide sequence that has at least about 85% sequence identity to the nucleotide sequence of SEQ ID NO: 8.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters, e.g., a hybrid promoter can have a nucleotide sequence that has at least about 85% sequence identity to the nucleotide sequence of SEQ ID NO: 10.
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 85% sequence identity to the nucleotide sequence of SEQ ID NO: 11.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 85% sequence identity to the nucleotide sequence of SEQ ID NO: 13.
  • the nucleic acid construct comprising the at least two promoters e.g. , a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 85% sequence identity to the nucleotide sequence of SEQ ID NO: 15.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters e.g.
  • a hybrid promoter can have a nucleotide sequence that has at least about 85% sequence identity to the nucleotide sequence of SEQ ID NO: 17.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter, can have a nucleotide sequence that has at least about 85% sequence identity to the nucleotide sequence of SEQ ID NO: 18.
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 90% sequence identity to the nucleotide sequence of SEQ ID NOs: 6-18.
  • the nucleic acid construct comprising the at least two promoters e.g. , a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 90% sequence identity to the nucleotide sequence of SEQ ID NO: 6.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters e.g.
  • a hybrid promoter can have a nucleotide sequence that has at least about 90% sequence identity to the nucleotide sequence of SEQ ID NO: 8.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters, e.g., a hybrid promoter can have a nucleotide sequence that has at least about 90% sequence identity to the nucleotide sequence of SEQ ID NO: 10.
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 90% sequence identity to the nucleotide sequence of SEQ ID NO: 11.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 90% sequence identity to the nucleotide sequence of SEQ ID NO: 13.
  • the nucleic acid construct comprising the at least two promoters e.g. , a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 90% sequence identity to the nucleotide sequence of SEQ ID NO: 15.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters e.g.
  • a hybrid promoter can have a nucleotide sequence that has at least about 90% sequence identity to the nucleotide sequence of SEQ ID NO: 17.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 95% sequence identity to the nucleotide sequence of SEQ ID NOs: 6-18.
  • the nucleic acid construct comprising the at least two promoters e.g. , a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 95% sequence identity to the nucleotide sequence of SEQ ID NO: 6.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters e.g.
  • a hybrid promoter can have a nucleotide sequence that has at least about 95% sequence identity to the nucleotide sequence of SEQ ID NO: 8.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters, e.g., a hybrid promoter can have a nucleotide sequence that has at least about 95% sequence identity to the nucleotide sequence of SEQ ID NO: 10.
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 95% sequence identity to the nucleotide sequence of SEQ ID NO: 11.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 95% sequence identity to the nucleotide sequence of SEQ ID NO: 13.
  • the nucleic acid construct comprising the at least two promoters e.g. , a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 95% sequence identity to the nucleotide sequence of SEQ ID NO: 15.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters e.g.
  • a hybrid promoter can have a nucleotide sequence that has at least about 95% sequence identity to the nucleotide sequence of SEQ ID NO: 17.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 96% sequence identity to the nucleotide sequence of SEQ ID NOs: 6-18.
  • the nucleic acid construct comprising the at least two promoters e.g. , a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 96% sequence identity to the nucleotide sequence of SEQ ID NO: 6.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters e.g.
  • a hybrid promoter can have a nucleotide sequence that has at least about 96% sequence identity to the nucleotide sequence of SEQ ID NO: 8.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters, e.g., a hybrid promoter can have a nucleotide sequence that has at least about 96% sequence identity to the nucleotide sequence of SEQ ID NO: 10.
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 96% sequence identity to the nucleotide sequence of SEQ ID NO: 11.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 96% sequence identity to the nucleotide sequence of SEQ ID NO: 13.
  • the nucleic acid construct comprising the at least two promoters e.g. , a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 96% sequence identity to the nucleotide sequence of SEQ ID NO: 15.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters e.g.
  • a hybrid promoter can have a nucleotide sequence that has at least about 96% sequence identity to the nucleotide sequence of SEQ ID NO: 17.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter, can have a nucleotide sequence that has at least about 96% sequence identity to the nucleotide sequence of SEQ ID NO: 18.
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 97% sequence identity to the nucleotide sequence of SEQ ID NOs: 6-18.
  • the nucleic acid construct comprising the at least two promoters e.g. , a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 97% sequence identity to the nucleotide sequence of SEQ ID NO: 6.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters e.g.
  • a hybrid promoter can have a nucleotide sequence that has at least about 97% sequence identity to the nucleotide sequence of SEQ ID NO: 8.
  • the nucleic acid construct comprising the at least two promoters e.g, a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters, e.g, a hybrid promoter can have a nucleotide sequence that has at least about 97% sequence identity to the nucleotide sequence of SEQ ID NO: 10.
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 97% sequence identity to the nucleotide sequence of SEQ ID NO: 11.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 97% sequence identity to the nucleotide sequence of SEQ ID NO: 13.
  • the nucleic acid construct comprising the at least two promoters e.g. , a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 97% sequence identity to the nucleotide sequence of SEQ ID NO: 15.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters e.g.
  • a hybrid promoter can have a nucleotide sequence that has at least about 97% sequence identity to the nucleotide sequence of SEQ ID NO: 17.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 98% sequence identity to the nucleotide sequence of SEQ ID NOs: 6-18.
  • the nucleic acid construct comprising the at least two promoters e.g. , a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 98% sequence identity to the nucleotide sequence of SEQ ID NO: 6.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters e.g.
  • a hybrid promoter can have a nucleotide sequence that has at least about 98% sequence identity to the nucleotide sequence of SEQ ID NO: 8.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters, e.g., a hybrid promoter can have a nucleotide sequence that has at least about 98% sequence identity to the nucleotide sequence of SEQ ID NO: 10.
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 98% sequence identity to the nucleotide sequence of SEQ ID NO: 11.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 98% sequence identity to the nucleotide sequence of SEQ ID NO: 13.
  • the nucleic acid construct comprising the at least two promoters e.g. , a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 98% sequence identity to the nucleotide sequence of SEQ ID NO: 15.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters e.g.
  • a hybrid promoter can have a nucleotide sequence that has at least about 98% sequence identity to the nucleotide sequence of SEQ ID NO: 17.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 99% sequence identity to the nucleotide sequence of SEQ ID NOs: 6-18.
  • the nucleic acid construct comprising the at least two promoters e.g. , a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 99% sequence identity to the nucleotide sequence of SEQ ID NO: 6.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters e.g.
  • a hybrid promoter can have a nucleotide sequence that has at least about 99% sequence identity to the nucleotide sequence of SEQ ID NO: 8.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters, e.g., a hybrid promoter can have a nucleotide sequence that has at least about 99% sequence identity to the nucleotide sequence of SEQ ID NO: 10.
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 99% sequence identity to the nucleotide sequence of SEQ ID NO: 11.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 99% sequence identity to the nucleotide sequence of SEQ ID NO: 13.
  • the nucleic acid construct comprising the at least two promoters e.g. , a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have a nucleotide sequence that has at least about 99% sequence identity to the nucleotide sequence of SEQ ID NO: 15.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters e.g.
  • a hybrid promoter can have a nucleotide sequence that has at least about 99% sequence identity to the nucleotide sequence of SEQ ID NO: 17.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters can have the nucleotide sequence of SEQ ID NOs: 6-18. In certain embodiments, the nucleic acid construct comprising the at least two promoters, e.g., a hybrid promoter, can have the nucleotide sequence of SEQ ID NOs: 7-18. In certain embodiments, the nucleic acid construct comprising the at least two promoters, e.g. , a hybrid promoter, can have the nucleotide sequence of SEQ ID NO: 6.
  • the nucleic acid construct comprising the at least two promoters can have the nucleotide sequence of SEQ ID NO: 7.
  • the nucleic acid construct comprising the at least two promoters, e.g., a hybrid promoter can have the nucleotide sequence of SEQ ID NO: 8.
  • the nucleic acid construct comprising the at least two promoters, e.g., a hybrid promoter can have the nucleotide sequence of SEQ ID NO: 9.
  • the nucleic acid construct comprising the at least two promoters can have the nucleotide sequence of SEQ ID NO: 10.
  • the nucleic acid construct comprising the at least two promoters can have the nucleotide sequence of SEQ ID NO: 11.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter
  • the nucleic acid construct comprising the at least two promoters e.g.
  • a hybrid promoter can have the nucleotide sequence of SEQ ID NO: 13.
  • the nucleic acid construct comprising the at least two promoters, e.g., a hybrid promoter can have the nucleotide sequence of SEQ ID NO: 14.
  • the nucleic acid construct comprising the at least two promoters, e.g., a hybrid promoter can have the nucleotide sequence of SEQ ID NO: 15.
  • the nucleic acid construct comprising the at least two promoters, e.g., a hybrid promoter can have the nucleotide sequence of SEQ ID NO: 16.
  • the nucleic acid construct comprising the at least two promoters can have the nucleotide sequence of SEQ ID NO: 17.
  • the nucleic acid construct comprising the at least two promoters e.g., a hybrid promoter, can have the nucleotide sequence of SEQ ID NO: 18.
  • the nucleic acid construct comprising the at least two promoters can include a first promoter comprising a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 4-5.
  • the nucleic acid construct comprising the at least two promoters e.g.
  • a hybrid promoter can include a second promoter comprising a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 1-3.
  • the nucleic acid construct comprising the at least two promoters can include a second promoter comprising a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 1.
  • the first promoter is a U6 promoter and the second promoter is a T7 promoter.
  • the nucleic acid construct comprising the at least two promoters can include (i) a first promoter comprising a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 4-5, and (ii) a second promoter comprising a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%,
  • the nucleic acid construct comprising the at least two promoters can include (i) a first promoter comprising a nucleotide sequence that has at least about 95% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 4-5, and (ii) a second promoter comprising a nucleotide sequence that has at least about 95% sequence identity to the nucleotide sequence of SEQ ID NO: 1.
  • the nucleic acid construct comprising the at least two promoters can include (i) a first promoter comprising a nucleotide sequence that has at least about 96% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 4-5, and (ii) a second promoter comprising a nucleotide sequence that has at least about 96% sequence identity to the nucleotide sequence of SEQ ID NO: 1.
  • the nucleic acid construct comprising the at least two promoters e.g.
  • a hybrid promoter can include (i) a first promoter comprising a nucleotide sequence that has at least about 97% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 4-5, and (ii) a second promoter comprising a nucleotide sequence that has at least about 97% sequence identity to the nucleotide sequence of SEQ ID NO: 1.
  • the nucleic acid construct comprising the at least two promoters can include (i) a first promoter comprising a nucleotide sequence that has at least about 98% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 4-5, and (ii) a second promoter comprising a nucleotide sequence that has at least about 98% sequence identity to the nucleotide sequence of SEQ ID NO: 1.
  • the nucleic acid construct comprising the at least two promoters can include (i) a first promoter comprising a nucleotide sequence that has at least about 99% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 4-5, and (ii) a second promoter comprising a nucleotide sequence that has at least about 99% sequence identity to the nucleotide sequence of SEQ ID NO: 1.
  • the nucleic acid construct comprising the at least two promoters can include (i) a first promoter comprising a nucleotide sequence that is identical to the nucleotide sequence of any one of SEQ ID NOs: 4-5, and (ii) a second promoter comprising a nucleotide sequence is identical to the nucleotide sequence of SEQ ID NO: 1.
  • the nucleic acid constructs, or compositions thereof, of the present disclosure can further comprise a nucleotide sequence (e.g., a polynucleotide) that encodes a nuclease.
  • a nucleotide sequence e.g., a polynucleotide
  • the nucleotide sequence that encodes a nuclease is operatively coupled to one or more of the at least two promoters.
  • the nucleotide sequence that encodes a nuclease is operatively coupled to a different promoter, e.g., a third promoter, present in the nucleic acid construct.
  • Non-limiting examples of nucleases comprise a zinc-finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN) and a Cas protein.
  • the nuclease is an RNA-guided nuclease.
  • the RNA-guided nuclease is a Cas protein.
  • Cas proteins are disclosed in Makarova and Koonin, Methods Mol. Biol. 1311 :47-75 (2015), the contents of which are incorporated herein by reference in their entirety.
  • Cas proteins comprise Casl, Cas2, Cas3, Cas3-HD, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Casl2a (Cpfl), Casl3, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, CsaX, Csm2, Csm3, Csm4, Csm5, Csm6, Csn2, Csbl, Csb2, Csb3, Csxl, Csx3, CsxlO, Csxl4, Csxl5, Csxl6, Csxl7, Csfl, Csf2, Csf3, Csf4, C2cl, C2c2 and C2c3.
  • the Cas protein is selected from the group consisting of a Cas9, a Cas 12a, a Cas 13 and a combination thereof. In certain embodiments, the Cas protein is a Cas9 protein. In certain embodiments, the Cas protein is a Casl2a protein.
  • the Cas protein is an engineered Cas protein that differs from a reference Cas protein, e.g., a wild-type Cas protein.
  • the reference Cas protein is a naturally occurring Cas protein.
  • the Cas protein comprises one or more amino acid variations compared to a reference Cas protein, e.g., a wild-type Cas protein.
  • an engineered Cas protein retains or substantially retains the nuclease (e.g., endonuclease) activity of the reference Cas protein.
  • the engineered Cas protein retains at least about 70%, about 80%, about 90%, about 95% or about 99% nuclease activity of the reference Cas protein.
  • an engineered Cas protein has no or no substantial cleavage activity.
  • a Cas protein can lack cleavage activity or have substantially less, e.g., less than 20%, about 10%, about 5% or about 1% of the cleavage activity of a reference Cas protein.
  • the engineered Cas protein comprises one or more deletions that reduces the size of the Cas protein while at least partially retaining the nuclease activity of the Cas protein.
  • the reduced size of the engineered Cas protein can allow flexibility with respect to the methods for delivering such engineered Cas proteins.
  • a Cas protein interacts with a gRNA molecule of the present disclosure and, in concert with the gRNA molecule, localizes to a target genomic sequence (e.g., a sequence that is complementary to the targeting domain sequence of the gRNA molecule) and a PAM sequence.
  • a target genomic sequence e.g., a sequence that is complementary to the targeting domain sequence of the gRNA molecule
  • PAM sequence e.g., the ability of a Cas protein to interact with and cleave a target genomic sequence.
  • cleavage of the target genomic sequence occurs upstream from the PAM sequence.
  • cleavage of the target genomic sequence occurs downstream from the PAM sequence.
  • Cas molecules from different species e.g., bacterial species, can recognize different PAM sequences.
  • a Cas protein for use in the present disclosure can be derived from any one of the following species: Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus thermophilus, Streptococcus agalactiae, Streptococcus parasanguinis, Streptococcus oralis, Streptococcus salivarius, Streptococcus macacae, Streptococcus dysgalactiae, Streptococcus anginosus, Streptococcus constellatus, Streptococcus pseudoporcinus, Streptococcus mutans, Listeria innocua, Spiroplasma apis, Spiroplasma syrphidicola, Porphyromonas catoniae, Prevotella intermedia, Treponema socranskii, Finegoldia magna, Pasteurella bettyae, Olivibacter sitiensis
  • a Cas protein for use in the present disclosure directs cleavage of one or both strands at a genomic location.
  • a Cas protein for use in the present disclosure directs cleavage of one or both strands within a genomic location.
  • the Cas protein directs cleavage of one or both strands within about 500 base pairs (e.g., within about 400, about 300, about 200, about 100, about 80, about 60, about 40, about 20, about 10 or about 5 base pairs) from the targeted genomic location.
  • a Cas protein e.g., a Cas9, that comprises functional RuvC and HNH nuclease domains can cleave both strands of a target nucleic acid sequence.
  • the Cas protein e.g., Cas9, comprises one functional endonuclease domain that allows the Cas protein to cleave only one strand (z.e., nick) of a target nucleic acid sequence.
  • a Cas9 nickase can comprise (i) a non-functional RuvC domain (e.g., a mutant RuvC domain) and (ii) a functional HNH domain (e.g., a wild type HNH domain).
  • a Cas9 nickase can comprise (i) a functional RuvC domain (e.g., wild type RuvC domain) and (ii) a nonfunctional HNH domain (e.g., a mutant HNH domain).
  • a Cas9 nickase comprises a functional HNH-like and comprise a mutation at D10, e.g., D10A.
  • a Cas9 nickase comprises a functional RuvC domain and comprises a mutation at H840, e.g., H840A. In certain embodiments, a Cas9 nickase comprises a functional RuvC domain and comprises a mutation at N863, e.g., N863A.
  • the nucleotide sequence encoding a Cas protein is codon optimized.
  • the nucleotide sequence encoding a Cas protein can be codon optimized, e.g., where at least one non-common codon or less- common codon has been replaced by a common codon, for optimized expression in a particular cell type, e.g., a mammalian cell.
  • the Cas protein is a fusion protein that comprises one or more heterologous protein domains.
  • a Cas fusion protein can comprise any additional protein domains, e.g., epitope tags, reporter sequences and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity.
  • the Cas protein comprises one or more nuclear localization sequences to promote accumulation of the Cas protein in a detectable amount in the nucleus of a cell.
  • Nuclear localization sequences are known in the art.
  • a Cas protein can comprise a nuclear localization sequence (e.g., from SV40) at its N-terminus and/or C-terminus.
  • a nucleic acid construct of the present disclosure can further comprise one or more unique sequences. In certain embodiments, a nucleic acid construct of the present disclosure can further comprise one or more unique sequences downstream of the target nucleic acid. In certain embodiments, a nucleic acid construct of the present disclosure can further comprise one or more unique sequences located 3’ to the target nucleic acid. For example, but not by way of limitation, a nucleic acid construct of the present disclosure can further comprise one or more barcodes. In certain embodiments, a nucleic acid construct of the present disclosure can further comprise two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more or ten barcodes.
  • the barcode can be under control of one or more of the promoters (e.g., hybrid promoter) comprised in a construct of the present disclosure. In certain embodiments, the barcode can be under control of a different promoter (e.g., a third or fourth promoter) comprised in a nucleic acid construct of the present disclosure. In certain embodiments, the barcode has a known nucleotide sequence. In certain embodiments, the barcode can be used as an identifier for an associated nucleic acid, e.g., target nucleic acid. Alternatively or additionally, the barcode can be used as an identifier of the source of an associated molecule, such as a cell-of-origin.
  • the promoters e.g., hybrid promoter
  • a different promoter e.g., a third or fourth promoter
  • the barcode has a known nucleotide sequence.
  • the barcode can be used as an identifier for an associated nucleic acid, e.g.,
  • the barcode is about 10 to about 50 nucleotides, e.g., about 10 to about 30 nucleotides, in length.
  • the barcode can be located downstream of the target nucleic acid in a nucleic acid construct of the present disclosure.
  • the barcode can be imaged using a method of the present disclosure.
  • a single target nucleic acid can be associated with a single barcode.
  • the present disclosure provides a plurality of constructs, where a first construct of the plurality of constructs can comprise a first target nucleic acid and a first barcode under control of a promoter disclosed herein and a second construct of the plurality of constructs can comprise a second target nucleic acid and a second barcode under control of a promoter disclosed herein.
  • the vector or delivery vehicle for a nucleic acid construct of the present disclosure is a viral vector (e.g., for generation of recombinant viruses).
  • the virus is a DNA virus (e.g., dsDNA or ssDNA virus).
  • the virus is an RNA virus (e.g., an ssRNA virus).
  • Exemplary viral vectors/viruses comprise, e.g., retroviruses, lentiviruses, adenovirus, adeno-associated virus (AAV), vaccinia viruses, poxviruses, and herpes simplex viruses.
  • the virus infects dividing and/or non-dividing cells.
  • the virus can integrate into the host genome. In certain embodiments, the virus is replication-competent. In certain embodiments, the virus is replication-defective, e.g., having one or more coding regions for the genes necessary for additional rounds of virion replication and/or packaging replaced with other genes or deleted.
  • a nucleic acid construct of the present disclosure is delivered by a non-vector-based method (e.g., using naked DNA or DNA complexes).
  • the DNA can be delivered, e.g., by electroporation, organically modified silica or silicate, transient cell compression or squeezing, gene gun, sonoporation, magnetofection, lipid-mediated transfection, dendrimers, inorganic nanoparticles, calcium phosphates, or a combination thereof.
  • the present disclosure provides a method for optical imaging target nucleic acids in different cell types.
  • in situ sequencing can be inefficient for optically detecting nucleic acids in a complex biological sample that comprise a variety of cell types.
  • the present disclosure provides an improved method for optical detecting target nucleic acids by in situ sequencing by increasing expression of the target nucleic acids after fixation.
  • cells that are to be analyzed by the methods of the present disclosure comprise one or more of the nucleic acid constructs disclosed herein or a vector comprising a nucleic acid construct disclosed herein. In certain embodiments, cells that are to be analyzed by the methods of the present disclosure comprise two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more or ten or more nucleic acid constructs described herein.
  • cells e.g., a plurality of cells
  • cells that are to be analyzed by the methods of the present disclosure can comprise at least one cell comprising a first nucleic acid construct and at least a second cell comprising a second nucleic acid construct, where the first and second nucleic acid constructs comprise different target nucleic acids.
  • a method of the present disclosure can comprise providing a cell or a plurality of cells that comprise at least one nucleic acid construct described herein.
  • a method of the present disclosure can comprise introducing at least one nucleic acid construct described herein into a cell or a plurality of cells.
  • a method of the present disclosure can comprise contacting a cell or a plurality of cells with at least one nucleic acid construct described herein (or a composition thereof).
  • the nucleic acid construct can be integrated into the genome of the cell or the genomes of the plurality of cells.
  • the cells e.g., the plurality of cells
  • the subject can be a human, non-human primate, e.g., an ape or a monkey, a farm animal, a mouse, a rat, a hamster, a guinea pig, a rabbit, a dog, cat, a sheep, a pig, a goat, a cow or a horse.
  • the subject is a human.
  • the cells can be obtained from a biological fluid.
  • biological fluids comprise whole blood, plasma, serum, sweat, urine, sputum, spinal fluid, pleural fluid, mucus, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal and genitourinary tracts, interstitial fluid, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, vaginal secretions, cerebrospinal fluid, intra-organ system fluid, ascitic fluid, tumor cyst fluid, amniotic fluid, bronchoalveolar fluid, biliary fluid and combinations thereof.
  • the cells can be obtained from a tissue, e.g., a tissue sample, or are present in a tissue sample. In certain embodiments, the cells (e.g., the plurality of cells) can be obtained from a tissue, e.g., a tissue sample. In certain embodiments, the cells (e.g., the plurality of cells) are present in a tissue, e.g., in a tissue sample.
  • Non-limiting examples of tissues comprise eye, muscle, skin, tendon, vein, artery, blood, heart, spleen, lymph node, bone, bone marrow, lung, bronchi, trachea, gut, small intestine, large intestine, colon, rectum, salivary gland, tongue, gallbladder, appendix, liver, pancreas, brain, stomach, skin, kidney, ureter, bladder, urethra, gonad, testicle, ovary, uterus, fallopian tube, thymus, pituitary, thyroid, adrenal or parathyroid tissue.
  • the cells can comprise primary cells, blood cells, somatic cells, epithelial cells, endothelial cells, fibroblast cells, microglia, neurons, astrocytes, cancer cells, cells derived from organoids or xenografts or stem cells, e.g., pluripotent stem cells (iPSCs) or embryonic stem cells.
  • the cells can comprise primary cells.
  • the cells for use in the present disclosure can be derived from stem cells, e.g., stem cells that have undergone natural differentiation or artificially induced reprogramming or transdifferentiation.
  • the cells (e.g., the plurality of cells) for use in the present disclosure can be fetal cells, e.g., obtained from or present in fetal tissue and/or amniotic fluid.
  • the methods of the present disclosure can be used to analyze fetal health and/or identify an abnormality in individual fetal cells.
  • the cells can be obtained from preserved samples, e.g., fixed samples, from frozen samples or from fresh samples, e.g., tissue samples.
  • the cells e.g., the plurality of cells
  • the cells are present in preserved samples, e.g., fixed samples, frozen samples or fresh samples, e.g., tissue samples.
  • the cells can be obtained from in vitro cell cultures.
  • the cells e.g., the plurality of cells
  • the cells can be immune cells.
  • immune cells comprise neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells (NK cells) and lymphocytes, e.g., B cells and T cells (e.g., cytotoxic T cells, natural killer T cells, regulatory T cells and helper T cells).
  • the cells can be modified immune cells that have been genetically engineered to express a chimeric antigen receptor (CAR), e.g., CAR T cells and CAR NK cells.
  • CAR chimeric antigen receptor
  • the cells can be obtained from and/or present in a malignancy of a tissue or a tumor.
  • malignancies comprise carcinomas, adenocarcinomas, sarcomas and fibroadenomas.
  • the cells are obtained from a cancer such as bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, head and neck cancer, kidney cancer, leukemia, lung cancer, lymphoma, melanoma, pancreatic cancer, parathyroid cancer, prostate cancer, stomach cancer, testicular cancer, thyroid cancer and uterine cancer.
  • the methods of the present disclosure can be used to identify mutations and/or gene alterations present in single cancer cells.
  • the cells can comprise diseased cells and healthy cells.
  • the cells can comprise cells obtained from a tumor or cancer and comprise non-cancerous cells (e.g., healthy cells that were located adjacent to the tumor or cancer or healthy cells obtained from a subject that does not have cancer).
  • the cells can be bacterial cells.
  • the methods of the present disclosure can be used to analyze the microbiome of a subject, e.g., the gut microbiome of a subject.
  • the cells can comprise cells having different developmental stages. In certain embodiments, the cells can comprise cells of different disease states.
  • the cells can be from any model organism.
  • the model organism can be E. coli, yeast, Arabidopsis, xenopus, zebrafish, drosophila melanogaster, ascidians, nematodes, mice and monkeys.
  • the cells can be cells that have been infected by an infectious agent.
  • infectious agents comprise viruses, bacteria, fungi and protozoans.
  • the cells e.g., the plurality of cells
  • the cells can be enriched for cells of interest to produce an enriched cell sample, which can be subjected to the methods of the present disclosure. Any technique known in the art can be used to enrich for the cells of interest.
  • the cells have been genetically modified to express and/or secrete an agent, e.g., a therapeutic agent.
  • an agent e.g., a therapeutic agent.
  • the cells to be used in the methods of the present disclosure express an antibody or an antibody fragment.
  • the cells for use in the present disclosure can be treated with an agent.
  • the agent can be a therapeutic agent.
  • methods of the present disclosure can be used in drug screening, e.g., to determine the genomic and/or transcriptional changes associated with a test therapeutic agent, e.g., a newly identified therapeutic agent.
  • methods of the present disclosure can be used in determining the genomic and/or transcriptional changes associated with resistance to a therapeutic agent.
  • Nonlimiting examples of such therapeutics comprise polypeptide therapeutics, e.g., antibodybased therapeutics, oligonucleotides, and small molecule therapeutics.
  • the therapeutic can be cell cycle regulators, kinase regulators (e.g., kinase inhibitors or activators), receptor regulators (e.g., receptor inhibitors or activators), chemotherapeutics and/or antibodies (e.g., agonist or antagonist antibodies).
  • kinase regulators e.g., kinase inhibitors or activators
  • receptor regulators e.g., receptor inhibitors or activators
  • chemotherapeutics and/or antibodies e.g., agonist or antagonist antibodies.
  • the present disclosure provides methods for imaging nucleic acids in a sample, e.g., in a plurality of cells. For example, but not by way of limitation, the present disclosure provides methods for imaging exogenous nucleic acids, e.g., exogenous RNAs, in a sample. In certain embodiments, the present disclosure provides methods for imaging one or more gRNAs in a sample. In certain embodiments, the present disclosure provides methods for imaging one or more barcodes in a sample.
  • methods of the present disclosure can comprise detecting a plurality of target nucleic acids in a plurality of cells, e.g., in a single sample.
  • the methods of the present disclosure can be used for a variety of purposes, e.g., the present disclosure provides methods for imaging the distribution of one or more nucleic acids in one or more cells in a plurality of cells, e.g., in a single sample.
  • methods of the present disclosure allow for visualizing a target nucleic acid in a plurality of cells and correlating the presence of the target nucleic acid to the characteristics of the cell containing the target nucleic acid.
  • the methods of the present disclosure can also be used to determine phenotypic characteristics of cells that express the target nucleic acid compared to cells that do not express the target nucleic acid or cells that express a different target nucleic acid.
  • the nucleic acid can also provide information reporting on the location of modified cells, enabling linkage of genotype to tissue localization of cells.
  • FIG. 1 and FIG. 9A provides a flowchart of an exemplary method of the present disclosure.
  • an exemplary method of the present disclosure can comprise one or more steps of: providing a sample, fixing the sample, permeabilizing the sample, decrosslinking the sample and performing an in vitro transcription process.
  • the method can further comprise performing an in situ sequencing process that comprises performing a reverse transcription process, performing a rolling circle amplification (RCA) process and performing sequencing by synthesis.
  • RCA rolling circle amplification
  • a method of the present disclosure can further include analyzing a change in a characteristic (e.g., a phenotypic characteristic) of one or more cells in the sample, e.g., prior to decrosslinking the sample and/or prior to performing an in vitro transcription process, as shown in FIG. 9A.
  • a characteristic e.g., a phenotypic characteristic
  • a method of the present disclosure can comprise providing a cell or a plurality of cells that comprise at least one nucleic acid construct described herein.
  • nucleic acid constructs and cells are described herein in Section II and III, respectively.
  • a method of the present disclosure can comprise introducing at least one nucleic acid construct described herein into a cell or a plurality of cells.
  • a method of the present disclosure can comprise contacting a cell or a plurality of cells with at least one nucleic acid construct described herein (or a composition thereof).
  • the plurality of cells can comprise at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 5,000, at least about 10,000, at least about 100,000, at least about 1,000,000, at least about 10,000,000, at least about 100,000,000 or at least about 1,000,000,000 cells.
  • the plurality of cells can comprise a variety of cells, e.g., cells of different types, cells of different lineages and/or cells with different genomic mutations.
  • the plurality of cells can comprise a variety of cell types pooled together.
  • a plurality of cells can comprise at least two or more cell types, at least three or more cell types, at least four or more cell types, at least five or more cell types, at least six or more cell types, at least seven or more cell types, at least eight or more cell types, at least nine or more cell types or at least ten or more cell types.
  • a method of the present disclosure can comprise providing a first cell or a first plurality of cells that comprise a first nucleic acid construct (e.g., that comprises a first target nucleic acid) and providing a second cell or a second plurality of cells that comprise a second nucleic acid construct (e.g., that comprises a second target nucleic acid).
  • a first nucleic acid construct e.g., that comprises a first target nucleic acid
  • second cell or a second plurality of cells that comprise a second nucleic acid construct (e.g., that comprises a second target nucleic acid).
  • a method of the present disclosure can comprise contacting a first cell or a first plurality of cells with a first nucleic acid construct described herein (or a composition thereof), e.g, that comprises a first target nucleic acid, and contacting a second cell or a second plurality of cells with a second nucleic acid construct described herein (or a composition thereof), e.g, that comprises a second target nucleic acid.
  • the first cell (or first plurality of cells) and the second cell (or second plurality of cells) can be pooled before undergoing sample preparation.
  • the first cell and the second cell can be different cell types or be the same cell type.
  • the method can further comprise incubating the cell or plurality of cells to promote expression of the target nucleic acid using the promoter (e.g., first promoter) that is active in live cells.
  • the promoter for expression in live cells e.g., first promoter
  • the promoter for expression in live cells is a Pol III or Pol II promoter.
  • the promoter for expression in live cells is a Pol III promoter.
  • the promoter for expression in live cells is a Pol II promoter.
  • the promoter that is active in live cells is a U6, U3, U2, U5, Hl, 75J, EF-la, CMV, tRNA, pGK, SV40, CAG, TRE, 7SK or VAI promoter.
  • the method comprises incubating (e.g., culturing) the cell or plurality of cells to express the target nucleic acid by using the Pol III promoter present in the nucleic acid construct present in the cell or plurality of cells.
  • the method comprises incubating (e.g., culturing) the cell or plurality of cells to express the target nucleic acid by using the Pol II promoter present in the nucleic acid construct present in the cell or plurality of cells. In certain embodiments, the method comprises incubating (e.g., culturing) the cell or plurality of cells to express the target nucleic acid by using the U6 promoter present in the nucleic acid construct present in the cell or plurality of cells.
  • the method can comprise incubating (e.g., culturing) the cell or plurality of cells to express the target nucleic acid from the promoter recognized by RNA polymerase III (e.g., a U6 promoter), e.g., the first promoter present with a hybrid promoter of the present disclosure.
  • RNA polymerase III e.g., a U6 promoter
  • the method can comprise incubating (e.g., culturing) the cell or plurality of cells for a sufficient amount of time to allow expression of the target nucleic acid, e.g., incubating (e.g., culturing) the cell or plurality of cells for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days or about 10 days.
  • the target nucleic acid encodes a gRNA
  • a method of the present disclosure can comprise incubating (e.g., culturing) the cell or plurality of cells to express the gRNA.
  • the method can comprise incubating (e.g., culturing) the cell or plurality of cells for a sufficient amount of time to allow expression of the target nucleic acid and/or editing of the target genomic sequence by the gRNA.
  • the method can comprise incubating (e.g., culturing) the cell or plurality of cells for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days or about 10 days to allow expression of the gRNA and/or editing of the target genomic sequence present in the cell by the gRNA.
  • the target nucleic acid includes a barcode
  • a method of the present disclosure can comprise incubating (e.g., culturing) the cell or plurality of cells to express the barcode.
  • the method can comprise incubating (e.g., culturing) the cell or plurality of cells for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days or about 10 days to allow expression of the barcode, e.g., allow expression of the target nucleic acid that includes the barcode.
  • the method can further comprise preparing the sample (e.g., a cell or a plurality of cells) for detection of the target nucleic acids.
  • exemplary methods of the present disclosure comprise the preparation of a sample (e.g., a cell or a plurality of cells) for the imaging of one or more target nucleic acids in the sample.
  • the sample e.g., the cell or plurality of cells
  • the sample preparation process can comprise a fixation process, a permeabilization process and/or a decrosslinking process.
  • sample preparation comprises a fixation process.
  • sample preparation can comprise a permeabilization process.
  • sample preparation can comprise a decrosslinking process.
  • sample preparation comprises a fixation process and a permeabilization process.
  • sample preparation comprises a fixation process and a decrosslinking process.
  • sample preparation comprises a fixation process, a permeabilization process and a decrosslinking process.
  • the fixation process comprises contacting a sample (e.g., a cell or a plurality of cells) with a fixative to generate a fixed sample (e.g., a fixed cell or a plurality of fixed cells).
  • fixatives comprise aldehydes (e.g., formaldehyde, paraformaldehyde and glutaraldehyde), imidoesters, N-Hydroxysuccinimide (NHS) esters (e.g., Bis-NHS ester), alcohols (e.g., methanol and ethanol), acetone and acetic acid.
  • the fixation process can be performed by exposing the sample to an aldehyde.
  • the fixation process can be performed by exposing the sample to formaldehyde. In certain embodiments, the fixation process can be performed by exposing the sample to glutaraldehyde. In certain embodiments, the fixation process can be performed by exposing the sample to a solution that comprises formaldehyde and glutaraldehyde. In certain embodiments, the fixation process can be performed by exposing the sample to a solution that comprises an aldehyde (e.g., formaldehyde) and acetic acid. In certain embodiments, the fixation process can be performed by exposing the sample to a solution that comprises a non-crosslinking fixative (e.g., an alcohol).
  • a non-crosslinking fixative e.g., an alcohol
  • the sample is fixed in a final fixative concentration (e.g., in v/v, w/w, v/w or w/v) of about 0.1% to about 10% , about 1% to about 10%, about 1% to about 8%, about 2% to about 7%, about 2% to about 6%, about 3% to about 6% or about 3% to about 5%.
  • a final fixative concentration e.g., in v/v, w/w, v/w or w/v
  • the sample is fixed in a final formaldehyde concentration (e.g., in v/v, w/w, v/w or w/v) of about 0.1% to about 10%, about 1% to about 10%, about 1% to about 8%, about 2% to about 7%, about 3% to about 6% or about 3% to about 5%, e.g., about 4%.
  • the sample is fixed in a final formaldehyde concentration of about 2% to about 6%.
  • the sample is fixed in a final formaldehyde concentration of about 4%.
  • the sample can be contacted with a fixative for about 5 hours or less, about 4 hours or less, about 3 hours or less, about 2 hours or less, about 1 hour or less, about 50 minutes or less, about 40 minutes or less, about 30 minutes or less, about 20 minutes or less, about 10 minutes or less or about 5 minutes or less.
  • the sample can be contacted with a fixative for about 1 hour or less.
  • the sample can be contacted with a fixative for about 5 minutes to about 1 hour, e.g., for about 5 minutes to about 30 minutes.
  • a sample can be contacted by a fixative at a temperature ranging from about -20°C to 50°C, e.g., at room temperature (RT).
  • RT room temperature
  • the sample can be fixed after the sample is contacted with a presently disclosed nucleic acid construct or composition thereof of the present disclosure but prior to detecting a target nucleic acid expressed from the nucleic acid construct in the sample.
  • the sample can be permeabilized after fixation.
  • the sample can be permeabilized after fixation of the sample and prior to detecting the target nucleic acid (e.g., to generate a fixed and permeabilized sample (e.g., a fixed and permeabilized cell or a plurality of fixed and permeabilized cells)).
  • a fixed and permeabilized sample e.g., a fixed and permeabilized cell or a plurality of fixed and permeabilized cells
  • Techniques for permeabilizing cells are known in the art and one of skill in the art would be able to assess the appropriateness of a particular technique for use in connection with the methods of the present disclosure.
  • Non-limiting examples of reagents for permeabilizing cells comprise detergents (e.g., saponin, Tween-20 and Triton X-100) and fixatives (e.g., acetone, methanol and ethanol).
  • the sample can be permeabilized with an alcohol, e.g., methanol, and/or a detergent, e.g., such as Triton X-100.
  • a reagent for permeabilization can be used at a concentration e.g.
  • v/v, w/w, v/w or w/v of about 0.1% to about 90%, e.g., about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 10% to about 80% or about 10% to about 80%.
  • a reagent for permeabilization can be used at a concentration of about 0.1% to about 90% v/v, e.g., about 10% to about 90% v/v, about 20% to about 90% v/v, about 30% to about 90% v/v, about 40% to about 90% v/v, about 50% to about 90% v/v, about 60% to about 90% v/v, about 10% to about 80% v/v or about 10% to about 80% v/v.
  • a reagent e.g., an alcohol
  • permeabilization can be performed by contacting the fixed sample about 70% v/v ethanol.
  • the sample e.g., fixed sample
  • the sample can be contacted with a permeabilization reagent for about 5 hours or less, about 4 hours or less, about 3 hours or less, about 2 hours or less, about 1 hour or less, about 50 minutes or less, about 40 minutes or less, about 30 minutes or less, about 20 minutes or less, about 10 minutes or less or about 5 minutes or less.
  • the sample, e.g., fixed sample can be contacted with a permeabilization reagent for about 1 hour or less.
  • the sample e.g., fixed sample
  • a permeabilization reagent for about 5 minutes to about 1 hour, e.g., for about 5 minutes to about 30 minutes.
  • a sample can be contacted with a permeabilization reagent, e.g., an alcohol, at a temperature ranging from about -80°C to 50°C, e.g., at room temperature (RT).
  • the permeabilization reagent comprises an alcohol.
  • the permeabilization reagent is ethanol (e.g., about 70% v/v ethanol) and/or methanol.
  • the permeabilization reagent comprises ethanol, e.g., about 70% v/v ethanol. In certain embodiments, the permeabilization reagent comprises methanol.
  • the sample can be permeabilized after the sample undergoes fixation but prior to detecting a target nucleic acid present in the nucleic acid construct in the sample. In certain embodiments, fixation and permeabilization can occur simultaneously.
  • the sample can be treated to remove crosslinking (referred to herein as decrosslinking), e.g., fixative-induced crosslinking.
  • the sample can undergo decrosslinking after fixation and permeabilization.
  • the sample can be decrosslinked after fixation and permeabilization of the sample and prior to detecting the target nucleic acid (e.g., to generate a fixed, permeabilized and decrosslinked sample (e.g., a fixed and permeabilized cell or a plurality of fixed, permeabilized and decrosslinked cells)).
  • the use of decrosslinking after fixation allows for an increase in the sensitivity and precision of target nucleic acid detection as shown in FIG.
  • decrosslinking allows for the allows for the use of additional types of fixatives, e.g., including aldehyde- based fixatives, prior to performing in vitro transcription.
  • decrosslinking comprises heating the fixed sample.
  • decrosslinking comprises contacting the sample with a reagent or composition thereof for decrosslinking.
  • a reagent or a composition thereof for decrosslinking comprises one or more salts, one or more small molecule catalysts, one or more buffers and/or one or more detergents.
  • the decrosslinking reagent or composition thereof comprises sodium chloride, a boronic acid or a derivative thereof (e.g., an aminophenylboronic acid or a cyclic boronic acid ester), a phosphonic acid ester, a bismuth salt (e.g., ranitidine bismuth citrate, colloidal bismuth subcitrate, tripotassium dicitratobismuthate, bismuth subsalicylate and bismuth subnitrate) and/or sodium bicarbonate.
  • a bismuth salt e.g., ranitidine bismuth citrate, colloidal bismuth subcitrate, tripotassium dicitratobismuthate, bismuth subsalicylate and bismuth subnitrate
  • sodium bicarbonate e.g., ranitidine bismuth citrate, colloidal bismuth subcitrate, tripotassium dicitratobismuthate, bismuth subsalicylate and bis
  • the sample e.g., fixed and permeabilized sample
  • a decrosslinking reagent or composition thereof for about 24 hours or less, e.g., about 22 hours or less, about 20 hours or less, about 18 hours or less, about 16 hours or less, about 14 hours or less, about 12 hours or less, about 10 hours or less, about 9 hours or less, about 8 hours or less, about 7 hours or less, about 6 hours or less, about 5 hours or less, about 4 hours or less, about 3 hours or less, about 2 hours or less or about 1 hour or less.
  • the sample e.g., fixed and permeabilized sample
  • the sample e.g., fixed and permeabilized sample
  • a decrosslinking reagent or composition thereof for about 2 hours to about 6 hours, e.g. , about 4 hours.
  • a sample can be contacted with a decrosslinking reagent or composition thereof, e.g, sodium chloride and/or sodium bicarbonate, at a temperature ranging from about 25°C to 100°C, e.g, at 65°C.
  • the sample can undergo decrosslinking after the sample undergoes fixation and permeabilization but prior to detecting a target nucleic acid present in the nucleic acid construct in the sample.
  • the analysis of a characteristic of the cells expressing (or not expressing) the target nucleic acid is performed prior to decrosslinking.
  • methods of the present disclosure comprise performing an in vitro transcription process.
  • methods of the present disclosure comprise synthesis of the nucleic acid that is contained within the nucleic acid construct present in the cell (e.g., in a plurality of cells) by in vitro transcription.
  • methods of the present disclosure comprise transcribing the target nucleic acid that is comprised within the nucleic acid construct present in the cell (e.g., in a plurality of cells) using the promoter that is active in fixed cells (e.g., the second promoter).
  • the method comprises transcribing the target nucleic acid using the second promoter, e.g., present within a hybrid promoter of the present disclosure.
  • in vitro transcription is performed by contacting the sample with a reagent composition that comprises the RNA polymerase that recognizes the promoter present in the nucleic acid construct for expressing the target nucleic acid in fixed cells (e.g., the second promoter).
  • the second promoter can be a T3 promoter, a T7 promoter and/or a Sp6 promoter.
  • the reagent composition comprises a phage RNA polymerase.
  • phage RNA polymerases comprise a bacteriophage T3 RNA polymerase, a bacteriophage T7 RNA polymerase, a bacteriophage SP6 RNA polymerase or a combination thereof.
  • the second promoter is a Sp6 promoter, and the RNA polymerase in the reagent composition is a Sp6 RNA polymerase.
  • the second promoter is a T3 promoter, and the RNA polymerase in the reagent composition is a T3 RNA polymerase.
  • the second promoter is a T7 promoter, and the RNA polymerase in the reagent composition is a T7 RNA polymerase.
  • the reagent composition can further comprise one or more of: NTPs, an RNase inhibitor and/or a buffer.
  • in vitro transcription can be performed in the presence of a reducing agent.
  • reducing agents are disclosed herein, e.g., DTT (dithiothreitol), DTE (dithioerythritol), L-glutathione (GSH) and TCEP (Tris (2- Carboxyethyl) phosphine hydrochloride).
  • DTT dithiothreitol
  • DTE dithioerythritol
  • GSH L-glutathione
  • TCEP Tris (2- Carboxyethyl) phosphine hydrochloride
  • in vitro transcription can be performed in the presence of DTT.
  • in vitro transcription can be performed in the presence of a reducing agent at a concentration from about 1 mM to about 50 mM.
  • in vitro transcription can be performed in the presence of a reducing agent at a concentration from about 1 mM to about 10 mM, e.g., about 5 mM.
  • a reducing agent at a concentration from about 1 mM to about 10 mM, e.g., about 5 mM.
  • the presence of DTT allows for the use of a reduced concentration of the T7 polymerase during in vitro transcription.
  • in vitro transcription is performed for about 4 hours to about 48 hours, e.g., about 24 hours, about 12 hours or about 6 hours. In certain embodiments, in vitro transcription is performed for about 24 hours. In certain embodiments, in vitro transcription is performed for about 12 hours. In certain embodiments, in vitro transcription is performed at a temperature ranging from about 25°C to 50°C, e.g., at 37°C. In certain embodiments, in vitro transcription is performed at about 37°C.
  • methods of the present disclosure can comprise the imaging of the one or more target nucleic acids in the sample (e.g., the cell or the plurality of cells).
  • methods of the present disclosure can further comprise the imaging of one target nucleic acid, two or more target nucleic acids, three or more target nucleic acids, four or more target nucleic acids, five or more target nucleic acids, six or more target nucleic acids, seven or more target nucleic acids, eight or more target nucleic acids, nine or more target nucleic acids or ten or more target nucleic acids in a sample.
  • methods of the present disclosure can comprise imaging a target nucleic acid in a cell of a sample (e.g., a plurality of cells) and imaging a second target nucleic acid in a second cell of the sample (e.g., the plurality of cells).
  • methods of the present disclosure can comprise imaging a target nucleic acid in a cell of a sample (e.g., a plurality of cells), imaging a second target nucleic acid in a second cell of the sample (e.g., the plurality of cells) and imaging a third target nucleic acid in a third cell of the sample (e.g., the plurality of cells).
  • methods of the present disclosure can comprise imaging a target nucleic acid in a subset of cells in the sample (e.g., a plurality of cells) and imaging a second target nucleic acid in a second subset of cells in the sample (e.g., the plurality of cells).
  • methods of the present disclosure can comprise imaging a target nucleic acid in a subset of cells in the sample (e.g., a plurality of cells), imaging a second target nucleic acid in a second subset of cells in the sample (e.g., the plurality of cells) and imaging a third target nucleic acid in a third subset of cells in the sample (e.g., the plurality of cells).
  • imaging of one or more target nucleic acids in the sample can comprise one or more of the following processes: performing a reverse transcription process, performing a gap filling process, performing an amplification process, performing a sequencing process and/or performing fluorescent in situ hybridization.
  • imaging one or more target nucleic acids in the sample can comprise one or more of the following processes: performing a reverse transcription process, performing a gap filling process, performing an amplification process and performing a sequencing process.
  • the amplified target nucleic acids are detected using in situ sequencing.
  • in situ sequencing is the sequencing of a nucleic acid directly in the cell and/or sample the nucleic acid is present in.
  • in situ sequencing allows for the resolution of target nucleic acids in single cells.
  • detection of one or more target nucleic acids in the sample can be performed using an in situ sequencing process that comprises performing a reverse transcription process, performing a gap filling process, performing an amplification process and performing a sequencing process.
  • methods of the present disclosure can comprise performing reverse transcription of the expressed target nucleic acid.
  • reverse transcription comprises the generation of cDNA from the target nucleic acid that is expressed from nucleic acid construct.
  • reverse transcription is performed by contacting the sample with a reagent composition that comprises a Reverse Transcriptase.
  • the reagent composition can further comprise one or more of: dNTPs, an RNase inhibitor, one or more primers and/or a buffer.
  • the one or more primers hybridizes to the target nucleic acid expressed from the nucleic acid construct, e.g., expressed from the first promoter, the second promoter or both promoters.
  • reverse transcription is performed for about 4 hours to about 48 hours, e.g., about 24 hours or about 12 hours. In certain embodiments, reverse transcription is performed for about 24 hours. In certain embodiments, reverse transcription is performed for about 12 hours. In certain embodiments, reverse transcription is performed at a temperature ranging from about 25°C to 50°C, e.g., at 37°C. In certain embodiments, reverse transcription is performed at about 37°C.
  • methods of the present disclosure can further comprise performing an amplification process to amplify the target nucleic acid, e.g., the cDNA generated from the reverse transcription process.
  • amplification process e.g., the cDNA generated from the reverse transcription process.
  • Suitable nucleic acid amplification methods known in the art can be assessed by those of skill in the art to identify strategies appropriate to amplify the target nucleic acid.
  • Non-limiting examples of such amplification processes comprise polymerase chain reaction (PCR), reverse transcriptase PCR, real-time PCR, rolling circle amplification (RCA), self-sustained sequence replication (3 SR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), transcription-mediated amplification (TMA), single primer isothermal amplification (SPIA), helicase-dependent amplification (HDA), loop mediated amplification (LAMP), recombinase-polymerase amplification (RPA), nicking enzyme amplification reaction (NEAR), nicking endonuclease assisted nanoparticle activation (NENNA) and ligase chain reaction (LCR).
  • PCR polymerase chain reaction
  • reverse transcriptase PCR real-time PCR
  • RCA rolling circle amplification
  • SR self-sustained sequence replication
  • NASBA nucleic acid sequence-based amplification
  • SDA strand displacement amplification
  • Fakruddin et al. J. Pharm. Bioallied. Sci. 5(4): 245-252 (2013) and Yan et al., Mol. BioSyst. 10:970-1003 (2014) disclose additional amplification processes for use in the present disclosure, the contents of each of which are disclosed in their entireties herein.
  • the amplification process can comprise a gap filling process.
  • a method of the present disclosure can comprise a gap filling process prior to amplification.
  • gap filling is performed by contacting the sample with a reagent composition that comprises a polymerase, e.g., a Taq polymerase, a padlock oligonucleotide and a ligase.
  • padlock oligonucleotides are linear oligonucleotides that can be converted into a circular DNA molecule by ligation upon hybridization to a target nucleic acid.
  • ligation is performed using a ligase that ligates single stranded DNA, e.g., Ampligase ligase.
  • a ligase that ligates single stranded DNA
  • hybridization of the padlock oligonucleotides to the target nucleic acids can occur in hybridization buffer for a period of about 1 hour to about 24 hours, e.g., from about 2 hours to about 20 hours, from about 2 hours to about 16 hours or from about 2 hours to about 12 hours.
  • the padlock oligonucleotides can be used at a concentration from about 100 pM to about 100 pM, e.g., about 100 pM to about 10 pM, about 100 pM to about 1,000 nM, about 100 pM to about 100 nM, about 100 pM to about 10 nM, about 100 pM to about 1 nM or about 1 nM to about 1,000 nM.
  • the padlock oligonucleotides can be used at a concentration from about 1 nM to about 1,000 nM, e.g., from about 1 nM to about 900 nM, from about 1 nM to about 800 nM, from about 1 nM to about 700 nM, from about 1 nM to about 600 nM, from about 1 nM to about 500 nM, from about 1 nM to about 400 nM, from about 1 nM to about 300 nM, from about 1 nM to about 200 nM, from about 10 nM to about 200 nM or from about 50 nM to about 200 nM.
  • the concentration of the padlock oligonucleotides is from about 1 nM to about 200 nM.
  • the gap filling process can occur for a period of about 1 hour to about 24 hours, e.g., from about 1 hours to about 20 hours, from about 10 minutes to about 16 hours, from about 1 hour to about 12 hours, from about 1 hour to about 10 hours, from about 1 hour to about 5 hours, from about 1 hour to about 2 hours.
  • the gap filling process can occur at a temperature ranging from about 10°C to 60°C, e.g., at 37°C and/or 45°C.
  • a plurality of oligonucleotides for detecting multiple different target nucleic acids can be used in the present disclosure.
  • each oligonucleotide e.g., padlock oligonucleotide, specifically binds to a single target nucleic acid.
  • the resulting circularized single-stranded DNA molecules can then be amplified using an amplification process.
  • performing an amplification process comprises contacting the sample with the reagents necessary for the amplification process and performing that process under conditions suitable for amplification of the target nucleic acid.
  • reagents comprise polymerases, nucleoside triphosphates or NTP analogues, primers, probes, primers, cofactors, ligation reaction reagents, endonucleases, lysis reagents, dyes, markers or labels.
  • additional reagents can comprise RNase inhibitors to protect the integrity of the RNA in the sample, e.g., by inhibiting the activity of RNase A, B and/or C.
  • the amplification reaction is RCA.
  • the amplification reaction comprises amplification reactions that use a polymerase with exonuclease activity.
  • the amplification reaction comprises amplification reactions that comprise phi29 polymerase.
  • the amplification process is an RCA process that uses phi29 polymerase. This RCA process produces a single-stranded DNA molecule, referred to herein as an “RCA amplicon,” containing multiple tandem repeats of the original target nucleic acid sequence.
  • the RCA process can be performed for about 24 hours or less, e.g., about 22 hours or less, about 20 hours or less, about 18 hours or less, about 16 hours or less, about 14 hours or less, about 12 hours or less, about 10 hours or less, about 9 hours or less, about 8 hours or less, about 7 hours or less, about 6 hours or less, about 5 hours or less, about 4 hours or less, about 3 hours or less, about 2 hours or less or about 1 hour or less.
  • the RCA process can be performed for about 1 hour to about 24 hours, e.g., about 2 hours to about 22 hours, about 4 hours to about 20 hours, about 6 hours to about 18 hours, about 8 hours to about 18 hours, about 10 hours to about 18 hours, about 12 hours to about 18 hours or about 14 hours to about 18 hours. In certain embodiments, the RCA process can be performed for about 16 hours. In certain embodiments, the RCA process can occur at a temperature ranging from about 10°C to 60°C, e.g., at 30°C. In certain embodiments, the RCA process can occur at a temperature of about 30°C.
  • the in situ sequencing technique can further comprise performing a sequencing process, e.g., a next-generation sequencing (NGS) process.
  • the sequencing process can be a sequencing by synthesis process.
  • the in situ sequencing technique can further comprise performing sequencing by synthesis.
  • amplicons generated by an amplification process are imaged using a sequencing process, e.g., a sequencing by synthesis process.
  • sequencing by synthesis relies on a primer complementary to a sequence present in the amplicons and a DNA polymerase to incorporate four reversible terminator-bound dNTPs.
  • each of the four reversible terminatorbound dNTPs are coupled to a different detectable label.
  • detectable labels comprise fluorescent labels (such as fluorescein (e.g., 5 -fluorescein, 6- carboxyfluorescein, 3’6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachloro- fluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, and the like), rhodamine, phycobiliproteins and R-phycoerythrin and quantum dots (e.g., zinc sulfide-capped cadmium selenide)), chromogenic labels, electron dense labels, chemiluminescent labels and radioactive labels.
  • fluorescent labels such as fluorescein (e.g., 5 -fluorescein, 6- carboxyfluorescein, 3’6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hex
  • each cycle has a duration of about 10 msec to about 500 msec, e.g., about 200 msec.
  • each cycle has a duration of about 10 msec to about 500 msec, e.g., about 200 msec.
  • multiple target nucleic acids within the same cell can be imaged by sequencing by synthesis.
  • different target nucleic acids present in different cells within the plurality of cells can be imaged using sequencing by synthesis.
  • a first cell within the plurality of cells can comprise a first nucleic acid that is imaged by sequencing by synthesis and a second cell within the plurality of cells can comprise a second nucleic acid that is imaged by sequencing by synthesis.
  • imaging of one or more target nucleic acids in the sample can comprise one or more of the following processes: performing a reverse transcription process, performing a gap filling process, performing an amplification process and performing fluorescent in situ hybridization.
  • imaging of one or more target nucleic acids in the sample can comprise performing fluorescent in situ hybridization, e.g., without performing a reverse transcription process, performing a gap filling process and/or performing an amplification process.
  • imaging of one or more target nucleic acids in the sample can comprise performing a reverse transcription process, performing an amplification process and performing fluorescent in situ hybridization.
  • imaging of one or more target nucleic acids in the sample can comprise performing a reverse transcription process and performing fluorescent in situ hybridization.
  • imaging of the target nucleic acids can be performed by in situ hybridization using detection probes.
  • a method of the present disclosure comprises an in situ sequencing process to image the target nucleic acids and further comprises imaging other nucleic acids within the plurality of cells using a detection probe.
  • a “detection probe” refers to an oligonucleotide that can selectively hybridize to at least a portion of a target sequence (e.g., a portion of a target sequence that has been amplified during RCA) under appropriate hybridization conditions.
  • the detection probe can comprise or consist of about 10 to about 50 nucleotides, e.g., about 15 to about 30 nucleotides.
  • a detection probe for use in the present disclosure comprises a sequence that specifically hybridizes to an RCA amplicon.
  • the detection probe is conjugated to a detectable label to facilitate imaging.
  • the detection probe is fluorescently labeled.
  • the detection probe covalently bound to a fluorescent label at its 5’ end or 3’ end.
  • specific detection probes e.g., detection probes for specific target nucleic acids
  • specific detection probes e.g., detection probes for specific target nucleic acids
  • each can be labeled with a different label, e.g., fluorophore, thus allowing for simultaneous imaging of a plurality of target nucleic acids.
  • a method of the present disclosure can further comprise analyzing the characteristics of the cells expressing (or not expressing) the target nucleic acid.
  • the present disclosure provides methods for analyzing one or more characteristics in a plurality of cells, e.g., in a plurality of cells comprising two or more different cell types, in a plurality of cells comprising cells having different developmental stages, in a plurality of cells comprising cells of different lineages, in a plurality of cells comprising cells of different disease states, in a plurality of cells comprising cells treated with an agent, in a plurality of cells comprising cells that are genetically modified, in a plurality of cells comprising cells obtained from a tissue sample and/or in a plurality of cells comprising cells obtained from a cell culture.
  • a method of the present disclosure can comprise analyzing a change in one or more characteristics of a cell that expresses a target nucleic acid, e.g., a gRNA, compared to a cell that does not express the target nucleic acid, e.g., the gRNA.
  • a target nucleic acid e.g., a gRNA
  • Non-limiting examples of characteristics that can be analyzed comprise cell viability, cell proliferation, cell size, cell morphology, cell motility, cell differentiation, cell adhesion, cell-cell contact, karyotype, chromosomal aberrations, nucleic acid expression levels (e.g., mRNA expression levels, e.g., RNA transcriptome), nucleic acid localization, protein expression levels, protein localization, nucleic acid modifications (e.g., methylation), post- translational modifications (e.g., phosphorylation, ubiquitination and/or glycosylation), activation of a cell signaling pathway, repression of a cell signaling pathway, enzymatic activity (e.g., enzymatic cleavage), chromatin accessibility, histone modifications and other epigenetic changes, concentrations of cytokines and hormones, drug sensitivity, drug absorption and metabolism pharmacokinetics and pharmacodynamics, and membrane potential.
  • nucleic acid expression levels e.g.,
  • the characteristic to be analyzed in a method of the present disclosure is the localization of one or more proteins. In certain embodiments, the characteristic to be analyzed in a method of the present disclosure is the expression, e.g., expression level, of one or more proteins.
  • a method of the present disclosure can include analyzing about 1 or more, about 10 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more or about 100 or more target proteins in a cell of the plurality of cells.
  • a method of the present disclosure can include analyzing about 1 to about 100 target proteins, e.g., about 10 to about 100 target proteins, about 20 to about 100 target proteins, about 30 to about 100 target proteins, about 40 to about 100 target proteins, about 50 to about 100 target proteins, about 60 to about 100 target proteins, about 70 to about 100 target proteins, about 80 to about 100 target proteins, about 90 to about 100 target proteins, about 1 to about 90 target proteins, about 1 to about 80 target proteins, about 1 to about 70 target proteins, about 1 to about 60 target proteins, about 1 to about 50 target proteins, about 1 to about 40 target proteins, about 1 to about 30 target proteins, about 1 to about 20 target proteins, about 1 to about 10 target proteins, about 10 to about 80 target proteins, about 10 to about 70 target proteins or about 10 to about 50 target proteins.
  • target proteins e.g., about 10 to about 100 target proteins, about 20 to about 100 target proteins, about 30 to about 100 target proteins, about 40 to about 100 target proteins, about 50 to about 100 target proteins, about 60 to about 100 target proteins, about 70 to
  • more than 1 target protein can be analyzed by immunofluorescence, e.g., by iterative immunolabeling (e.g., indirect or direct) and removal (e.g., by chemical bleaching) for highly multiplexed imaging of proteins.
  • the immunofluorescence process can be an iterative bleaching extends multiplexity (IBEX) process or an iterative indirect immunofluorescence imaging (4i) process.
  • a method of the present disclosure can comprise analyzing a change in protein expression levels, e.g., as shown in FIG. 11F and FIG. 11J.
  • a method of the present disclosure can further include performing immunofluorescence to detect a change in expression of one or more target proteins, e.g. two or more, three or more, four or more or five or more target proteins.
  • a method of the present disclosure can further include performing immunofluorescence to detect a change in expression of about 1 or more, about 10 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more or about 100 or more target proteins.
  • a method of the present disclosure can comprise analyzing a change in protein localization, e.g, as shown in FIG. 11F and FIG. 11 J.
  • a method of the present disclosure can further include performing immunofluorescence to detect a change in localization of one or more target proteins, e.g., two or more, three or more, four or more or five or more target proteins.
  • a method of the present disclosure can further include performing immunofluorescence to detect a change in localization of about 1 or more, about 10 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more or about 100 or more target proteins.
  • the characteristic to be analyzed in a method of the present disclosure is the localization of one or more nucleic acids, e.g., mRNAs. In certain embodiments, the characteristic to be analyzed in a method of the present disclosure is the expression, e.g., expression level, of one or more nucleic acids, e.g., mRNAs.
  • a method of the present disclosure can include analyzing about 1 or more, about 10 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more, about 100 or more, about 200 or more, about 300 or more, about 400 or more, about 500 or more, about 600 or more, about 700 or more, about 800 or more, about 900 or more, about 1,000 or more, about 1,500 or more, about 2,000 or more, about 2,500 or more or about 3,000 or more nucleic acids, e.g., mRNAs, in a cell of the plurality of cells.
  • nucleic acids e.g., mRNAs
  • a method of the present disclosure can include analyzing about 1 to about 2,000, about 1 to about 1,500, about 1 to about 1,000, about 1 to about 500, about 1 to about 100, about 10 to about 3,000, about 50 to about 3,000, about 100 to about 3,000, about 500 to about 3,000, about 1,000 to about 3,000, about 1,500 to about 3,000, about 2,000 to about 3,000, about 10 to about 1,000, about 10 to about 500 or about 10 to about 100 more nucleic acids, e.g., mRNAs, in a cell of the plurality of cells.
  • the nucleic acids being analyzed are distinct from the target nucleic acids included in the nucleic acid construct present within the cells.
  • a method of the present disclosure can comprise analyzing a change in nucleic acid expression levels (e.g., mRNA expression levels, e.g., RNA transcriptome).
  • a method of the present disclosure can include performing in situ hybridization, e.g., fluorescent in situ hybridization (FISH), for analyzing a change in nucleic acid expression levels (e.g., mRNA expression levels, e.g., RNA transcriptome).
  • FISH fluorescent in situ hybridization
  • a method of the present disclosure can comprise analyzing a change in the expression levels of about 1 or more, about 10 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more, about 100 or more, about 200 or more, about 300 or more, about 400 or more, about 500 or more, about 600 or more, about 700 or more, about 800 or more, about 900 or more, about 1,000 or more, about 1,500 or more, about 2,000 or more, about 2,500 or more or about 3,000 or more nucleic acids (e.g., mRNAs).
  • nucleic acids e.g., mRNAs
  • a method of the present disclosure can comprise analyzing a change in nucleic acid localization (e.g., mRNA localization), e.g., as shown in FIG. 11F, FIG. 11 J and FIG. 13N.
  • a method of the present disclosure can include performing in situ hybridization, e.g., fluorescent in situ hybridization (FISH), for analyzing a change in nucleic acid localization (e.g., mRNA localization).
  • FISH fluorescent in situ hybridization
  • a method of the present disclosure can comprise analyzing a change in the localization of about 1 or more, about 10 or more, about 20 or more, about 30 or more, about 40 or more, about 50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more, about 100 or more, about 200 or more, about 300 or more, about 400 or more, about 500 or more, about 600 or more, about 700 or more, about 800 or more, about 900 or more, about 1,000 or more, about 1,500 or more, about 2,000 or more, about 2,500 or more or about 3,000 or more nucleic acids (e.g., mRNAs).
  • nucleic acids e.g., mRNAs
  • a method of the present disclosure can comprise analyzing the characteristics of a cell expressing the target nucleic acid compared to a cell that does not express the target nucleic acid to determine a change in a characteristic that is associated with the expression of the target nucleic acid.
  • the method comprises providing a plurality of cells, wherein at least one cell of the plurality of cells comprises a nucleic acid construct comprising a first promoter, a second promoter and a target nucleic acid, wherein the first promoter and the second promoter are located upstream to the target nucleic acid in the nucleic acid construct.
  • the nucleotide sequence of the second promoter is incorporated into the nucleotide sequence of the first promoter.
  • the method can further comprise culturing the plurality of cells to allow expression of the target nucleic acid using the first promoter, fixing the plurality of cells to generate a plurality of fixed cells and transcribing the target nucleic acid in at least one cell of the plurality of fixed cells using the second promoter.
  • the method comprises performing an amplification process for amplifying the target nucleic acid, imaging the amplified target nucleic acid using in situ sequencing (e.g., sequencing by synthesis) and analyzing a change in a characteristic of one or more cells of the plurality of cells associated with expression of the target nucleic acid.
  • the change in a characteristic is determined prior to the imaging of the amplified target nucleic acid (e.g., by in situ sequencing).
  • the method can further include decrosslinking the fixed cells prior to transcribing the target nucleic acid in the plurality of fixed cells using the second promoter.
  • a method of the present disclosure can comprise analyzing the characteristics of a cell expressing the target nucleic acid compared to a cell that does not express the target nucleic acid to determine a change in a characteristic that is associated with the expression of the target nucleic acid.
  • the method comprises providing a plurality of cells, wherein at least one cell of the plurality of cells comprises a nucleic acid construct comprising a first promoter, a second promoter and a target nucleic acid, wherein the first promoter and the second promoter are located upstream to the target nucleic acid in the nucleic acid construct.
  • the nucleotide sequence of the second promoter is incorporated into the nucleotide sequence of the first promoter.
  • the method can further comprise culturing the plurality of cells to allow expression of the target nucleic acid using the first promoter and fixing the plurality of cells to generate a plurality of fixed cells.
  • the method can further include analyzing a change in a characteristic of one or more cells of the plurality of cells associated with expression of the target nucleic acid.
  • the characteristic can be protein and/or RNA expression levels or localization.
  • the method includes transcribing the target nucleic acid in at least one cell of the plurality of fixed cells using the second promoter.
  • the method comprises performing an amplification process for amplifying the target nucleic acid and imaging the amplified target nucleic acid using in situ sequencing (e.g., sequencing by synthesis).
  • the method includes correlating the change in characteristic with the expression of the target nucleic acid as determined by in situ sequencing.
  • the method can further include decrosslinking the fixed cells prior to transcribing the target nucleic acid in the plurality of fixed cells using the second promoter.
  • a method of the present disclosure comprises providing a plurality of cells, wherein at least one cell of the plurality of cells comprises a first nucleic acid construct and at least a second cell comprises a second nucleic acid construct.
  • the first nucleic acid construct comprises a first promoter, a second promoter and a first target nucleic acid, wherein the first promoter and the second promoter are located upstream to the first target nucleic acid in the first nucleic acid construct.
  • the second nucleic acid construct comprises the first promoter, the second promoter and a second target nucleic acid, wherein the first promoter and the second promoter are located upstream to the second target nucleic acid in the second nucleic acid construct.
  • the nucleotide sequence of the second promoter is incorporated into the nucleotide sequence of the first promoter.
  • the method can further comprise culturing the plurality of cells to allow expression of the first and second target nucleic acids using the first promoter, fixing the plurality of cells to generate a plurality of fixed cells and transcribing the first and second target nucleic acids in the plurality of fixed cells using the second promoter.
  • the genome screen comprises performing an amplification process for amplifying the first and second target nucleic acids, imaging the amplified target nucleic acids using in situ sequencing (e.g., sequencing by synthesis) and analyzing a change in a characteristic of one or more cells of the plurality of cells associated with expression of the first target nucleic acid and/or second target nucleic acid.
  • the change in a characteristic is determined prior to the imaging of the amplified target nucleic acid (e.g., by in situ sequencing).
  • the method can further include decrosslinking the fixed cells prior to transcribing the first and second target nucleic acids in the plurality of fixed cells using the second promoter.
  • a method of the present disclosure can comprise analyzing the characteristics of a cell expressing a gRNA compared to a cell that does not express the gRNA to determine a change in a characteristic that is associated with the genetic perturbation caused by the gRNA, e.g., the knock down or knock out of the gene targeted by the gRNA.
  • the present disclosure provides a method for performing a genomic screen.
  • a genomic screen of the present disclosure comprises providing a plurality of cells, wherein at least one cell of the plurality of cells comprises a nucleic acid construct comprising a first promoter, a second promoter and a target nucleic acid encoding a gRNA, wherein the first promoter and the second promoter are located upstream to the target nucleic acid in the nucleic acid construct.
  • the nucleotide sequence of the second promoter is incorporated into the nucleotide sequence of the first promoter.
  • the genomic screen can further comprise culturing the plurality of cells to allow expression of the gRNA using the first promoter, fixing the plurality of cells to generate a plurality of fixed cells and transcribing the gRNA in at least one cell of the plurality of fixed cells using the second promoter.
  • the genomic screen comprises performing an amplification process for amplifying the gRNA, imaging the amplified gRNA in situ sequencing (e.g., using sequencing by synthesis) and analyzing a change in a characteristic of one or more cells of the plurality of cells associated with expression of the gRNA.
  • the change in a characteristic is determined prior to the imaging of the amplified target nucleic acid (e.g., by in situ sequencing).
  • the method can further include decrosslinking the fixed cells prior to transcribing the gRNA in the plurality of fixed cells using the second promoter.
  • a genomic screen of the present disclosure comprises providing a plurality of cells, wherein at least one cell of the plurality of cells comprises a first nucleic acid construct and at least a second cell comprises a second nucleic acid construct.
  • the first nucleic acid construct comprises a first promoter, a second promoter and a first target nucleic acid encoding a first gRNA, wherein the first promoter and the second promoter are located upstream to the first target nucleic acid in the first nucleic acid construct.
  • the second nucleic acid construct comprises the first promoter, the second promoter and a second target nucleic acid encoding a second gRNA, wherein the first promoter and the second promoter are located upstream to the second target nucleic acid in the second nucleic acid construct.
  • the nucleotide sequence of the second promoter is incorporated into the nucleotide sequence of the first promoter.
  • the genome screen can further comprise culturing the plurality of cells to allow expression of the first and second gRNA using the first promoter, fixing the plurality of cells to generate a plurality of fixed cells and transcribing the first and second gRNA in the plurality of fixed cells using the second promoter.
  • the genome screen comprises performing an amplification process for amplifying the first and second gRNAs, imaging the amplified gRNAs using in situ sequencing (e.g., sequencing by synthesis) and analyzing a change in a characteristic of one or more cells of the plurality of cells associated with expression of the first gRNA and/or second gRNA.
  • the change in a characteristic is determined prior to the imaging of the amplified target nucleic acid (e.g., by in situ sequencing).
  • the method can further include decrosslinking the fixed cells prior to transcribing the first and second gRNAs in the plurality of fixed cells using the second promoter.
  • the barcode can be imaged by in situ sequencing in a method of the present disclosure.
  • a method of the present disclosure can include providing a plurality of cells, wherein at least one cell of the plurality of cells comprises a nucleic acid construct comprising a first promoter, a second promoter, a target nucleic acid and a barcode, wherein the first promoter and the second promoter are located upstream to the target nucleic acid and the barcode in the nucleic acid construct.
  • the nucleotide sequence of the second promoter is incorporated into the nucleotide sequence of the first promoter.
  • the method can further comprise culturing the plurality of cells to allow expression of the target nucleic acid using the first promoter, fixing the plurality of cells to generate a plurality of fixed cells and transcribing the target nucleic acid and the barcode in at least one cell of the plurality of fixed cells using the second promoter.
  • the method comprises performing an amplification process for amplifying the target nucleic acid and/or the barcode, imaging the amplified target nucleic acid and/or the barcode using in situ sequencing and analyzing a change in a characteristic of one or more cells of the plurality of cells associated with expression of the target nucleic acid.
  • only the barcode is imaged using in situ sequencing.
  • the change in a characteristic is determined prior to the imaging of the amplified target nucleic acid and/or barcode (e.g., by in situ sequencing).
  • the method can further include decrosslinking the fixed cells prior to transcribing the target nucleic acid and barcode in the plurality of fixed cells using the second promoter.
  • a method of the present disclosure can comprise analyzing the characteristics of a cell expressing the target nucleic acid compared to a cell that does not express the target nucleic acid to determine a change in a characteristic that is associated with the expression of the target nucleic acid.
  • the nucleic acid construct further comprises a barcode
  • the barcode can be imaged by in situ sequencing in a method of the present disclosure and the presence of the barcode confirms the presence of the target nucleic acid in the cell.
  • the method of the present disclosure can include providing a plurality of cells, wherein at least one cell of the plurality of cells comprises a nucleic acid construct comprising a first promoter, a second promoter, a target nucleic acid and a barcode, wherein the first promoter and the second promoter are located upstream to the target nucleic acid and the barcode in the nucleic acid construct.
  • the nucleotide sequence of the second promoter is incorporated into the nucleotide sequence of the first promoter.
  • the method can further comprise culturing the plurality of cells to allow expression of the target nucleic acid using the first promoter and fixing the plurality of cells to generate a plurality of fixed cells.
  • the method can further include analyzing a change in a characteristic of one or more cells of the plurality of cells associated with expression of the target nucleic acid.
  • the characteristic can be protein and/or RNA expression levels or localization.
  • the method can further include transcribing the target nucleic acid and the barcode in at least one cell of the plurality of fixed cells using the second promoter.
  • the method comprises performing an amplification process for amplifying the target nucleic acid and/or the barcode, imaging the amplified target nucleic acid and/or the barcode using in situ sequencing and analyzing a change in a characteristic of one or more cells of the plurality of cells associated with expression of the target nucleic acid.
  • only the barcode is imaged using in situ sequencing.
  • the presence of the barcode confirms the presence of the target nucleic acid in the cell.
  • the method can further include decrosslinking the fixed cells prior to transcribing the target nucleic acid and barcode in the plurality of fixed cells using the second promoter.
  • methods of the present disclosure can comprise staining the cells with other imaging agents.
  • methods of the present disclosure can comprise performing a cell painting technique and using immunofluorescence.
  • additional imaging agents can be used to label cellular structures including, but not limited to, nuclei, cytoskeleton, golgi, endoplasmic reticulum, mitochondria and cell membranes.
  • a method of the present disclosure can further comprise labeling the nucleic of the cells within the sample, e.g., the plurality of cells.
  • a method of the present disclosure can further comprise analyzing protein expression and/or localization of a cell expressing or not expressing the target nucleic acid.
  • methods of the present disclosure can comprise detecting one or more proteins in the sample (e.g., cell or plurality of cells).
  • methods of the present disclosure can comprise the staining and imaging of one or more target proteins, e.g., about 1 to about 100 target proteins.
  • methods of the present disclosure can comprise the staining and imaging of two or more target proteins, three or more target proteins, four or more target proteins, five or more target proteins, six or more target proteins, seven or more target proteins, eight or more target proteins, nine or more target proteins or ten or more target proteins.
  • proteins that can be stained and imaged using the methods of the present disclosure comprise any protein that is present in or on the surface of a cell.
  • the target protein can be an intracellular protein, an extracellular protein or a transmembrane protein.
  • the target protein is a mutated form of a protein or a wild type form of a protein.
  • the target protein is an exogenous protein.
  • the target protein is an endogenous protein.
  • a method of the present disclosure includes performing immunofluorescence for detecting one or more target proteins, e.g., two or more target proteins, three or more target proteins, four or more target proteins, five or more target proteins, six or more target proteins, seven or more target proteins, eight or more target proteins, nine or more target proteins or ten or more target proteins.
  • a method of the present disclosure can further comprise analyzing expression of other nucleic acids, e.g., mRNA expression, in a cell expressing or not expressing the target nucleic acid.
  • methods of the present disclosure can comprise detecting one or more nucleic acids (other than the target nucleic acids) in the sample (e.g., cell or plurality of cells). In certain embodiments, about 1 to about 2,000 nucleic acids (other than the target nucleic acids) can be detected in a sample.
  • methods of the present disclosure can comprise performing transcriptomics to identify the expression level of mRNAs in cells expressing or not expressing the target nucleic acid.
  • the staining and imaging of one or more protein targets in a sample can comprise contacting a sample with a reagent that binds to a target protein, also referred to herein as a “protein binding reagent,” in the sample.
  • the protein binding reagent is a reagent that specifically binds to a target protein, e.g., specifically binds to a target protein of a cell in a sample.
  • the reagent that bind to the target protein allows for the imaging of the target protein.
  • the reagent that binds to the target protein allows for the quantitative analysis of the target protein.
  • Non-limiting example of protein binding reagents comprise antibodies or antibody binding fragments thereof, aptamers, peptides and small molecules.
  • the sample can be contacted with the protein binding reagent, e.g., the antibody specific for the target protein, for amount of time and under conditions to support specific binding of the protein binding reagent to the target protein.
  • analyzing a change in one or more characteristics of a cell that expresses a target nucleic acid occurs after fixation of the plurality of cells and prior to transcription, e.g., as shown in FIG. 9A.
  • a method of the present disclosure e.g., a method for analyzing one or more characteristics of a plurality of cells, can include (a) providing a plurality of cells, where at least one cell of the plurality of cells comprises a nucleic acid construct comprising a first promoter, a second promoter and a target nucleic acid, where the first promoter and the second promoter are located upstream to the target nucleic acid in the nucleic acid construct, (b) culturing the plurality of cells to allow expression of the target nucleic acid using the first promoter, (c) fixing the plurality of cells to generate a plurality of fixed cells, (d) analyzing a change in one or more characteristics of the cell of the plurality of fixed cells,
  • a method of the present disclosure e.g., a method for analyzing one or more characteristics of a plurality of cells, can include (a) providing a plurality of cells, where at least one cell of the plurality of cells comprises a nucleic acid construct comprising a first promoter, a second promoter and a target nucleic acid, where the first promoter and the second promoter are located upstream to the target nucleic acid in the nucleic acid construct, (b) culturing the plurality of cells to allow expression of the target nucleic acid using the first promoter, (c) fixing the plurality of cells to generate a plurality of fixed cells, (d) analyzing a change in one or more characteristics of the cell of the plurality of fixed cells, (e) decrosslinking the cells to generate a plurality of decrosslinked cells, (f) transcribing the target nucleic acid in the at least one cell of the decrosslinked cells using the second promoter, (g) performing an amplification process to ampli
  • kits for performing the methods of the present disclosure provides kits for performing the methods of the present disclosure.
  • the present disclosure provides kits containing materials for performing a method for optically imaging a nucleic acid in a sample.
  • a kit of the present disclosure can comprise one or more nucleic acid constructs described herein, e.g., two or more, three or more, four or more or five or more nucleic acid constructs.
  • a kit of the present disclosure can comprise a composition comprising one or more nucleic acid constructs described herein.
  • a kit of the present disclosure can comprise a vector comprising one or more nucleic acid constructs described herein.
  • a kit of the present disclosure can comprise a nucleic acid construct that comprises at least two promoter sequences upstream of the target nucleic acid, e.g., gRNA nucleotide sequence.
  • the first promoter is a promoter for expressing the target nucleic acid in live cells and the second promoter is a promoter for expressing the target nucleic acids in fixed cells.
  • the first promoter is a U6 promoter and the second promoter is a T7 promoter.
  • kits of the present disclosure can further comprise reagents for preparing sample.
  • a kit of the present disclosure can comprise reagents for performing a fixation process, a permeabilization process and/or a decrosslinking process.
  • reagents comprise a fixative (e.g., an aldehyde), a permeabilizing reagent (e.g., an alcohol) and/or a decrosslinking reagent (e.g., sodium chloride and/or sodium bicarbonate).
  • a kit of the present disclosure can further comprise reagents for performing an in vitro transcription reaction, e.g., an in vitro transcription reaction.
  • a kit of the present disclosure can further comprise reagents for performing a reverse transcription reaction.
  • a kit of the present disclosure can further comprise reagents for performing an amplification reaction, e.g., an RCA reaction.
  • a kit of the present disclosure can further comprise reagents for performing a sequencing by synthesis reaction.
  • a kit of the present disclosure can comprise reagents comprising one more of the following: polymerases (e.g., phi29 polymerase and/or Taq polymerase), RNA polymerases (e.g., T7 RNA polymerase), reverse transcriptases, nucleoside triphosphates or NTP analogues, primers, padlock probes, cofactors, ligation reaction reagents, endonucleases, lysis reagents, dyes, markers, RNase inhibitors and labels.
  • polymerases e.g., phi29 polymerase and/or Taq polymerase
  • RNA polymerases e.g., T7 RNA polymerase
  • reverse transcriptases e.g., reverse transcriptases
  • nucleoside triphosphates or NTP analogues e.g., primers, padlock probes, cofactors, ligation reaction reagents, endonucleases, lysis reagents
  • a kit of the present disclosure can include one or more reducing agents, e.g., for performing an in vitro transcription reaction.
  • reducing agents include DTT (dithiothreitol), DTE (dithioerythritol), L- glutathione (GSH) and TCEP (Tris (2-Carboxyethyl) phosphine hydrochloride).
  • a kit of the present disclosure includes DTT.
  • a kit of the present disclosure can include DTT in a container at a concentration from about 1 mM to about 50 mM, e.g., about 5 mM.
  • a kit of the present disclosure can comprise one or more detection probes, e.g., fluorescently labeled detection probes.
  • suitable containers comprise bottles, test tubes, vials and microtiter plates.
  • the containers can be formed from a variety of materials such as glass or plastic.
  • kits of the present disclosure can include a buffer, e.g., a buffer for performing an in vitro transcription reaction, comprising one or more reducing agents, e.g., DTT.
  • a buffer e.g., a buffer for performing an in vitro transcription reaction, comprising one or more reducing agents, e.g., DTT.
  • the kit further comprises a package insert that provides instructions for using the components provided in the kit.
  • a kit of the present disclosure can comprise a package insert that provides instructions for performing methods for imaging one or more target nucleic acids in a single sample.
  • the components of the kit are provided in predetermined ratios, with the relative amounts of the various reagents suitably varied to obtain the desired sensitivity and throughput of the disclosed methods.
  • the present disclosure provides systems for performing the methods of the present disclosure.
  • the present disclosure provides systems containing materials for performing a method for optically imaging a nucleic acid in a sample.
  • the present disclosure provides systems that include materials or reagents (e.g., in one or more reservoirs or containers) for performing a method for optically imaging a nucleic acid in a sample.
  • the system is an automated system.
  • the automated system includes one or more automated pipettes for dispensing materials or reagents onto a sample.
  • a system of the present disclosure can include a Xenium analyzer and/or Xenium slides for use in a Xenium analyzer (lOx Genomics), e.g., as shown in FIG. 13.
  • an automated system can include a Xenium analyzer and/or Xenium slides.
  • a system of the present disclosure can include a Xenium analyzer for performing a method disclosed herein, where the cells analyzed using the disclosed methods and systems are cells obtained from a cell culture.
  • a system of the present disclosure can include a Xenium analyzer for performing a method disclosed herein, where the cells analyzed using the disclosed methods and systems are cells in a tissue sample (e.g., a tissue section).
  • the cells to be analyzed e.g., cells containing a nucleic acid construct of the present disclosure
  • the cells to be analyzed are placed on Xenium slides and analyzed on the Xenium analyzer prior to performing one or more steps of a method of the present disclosure.
  • a method of the present disclosure includes the analysis of the transcriptome (e.g., RNA localization) of a sample (e.g., dissociated cells from a cell culture or a tissue sample) on a Xenium slide using a Xenium device followed by performing one or more steps of a method of the present disclosure, e.g., imaging of the target nucleic acid (e.g., by in situ sequencing), on a separate device.
  • the Xenium slide that is used with the Xenium device for performing transcriptomics can be transferred to a separate device (e.g., imaging device) for performing imaging of the target nucleic acid (e.g., by in situ sequencing).
  • the data obtained from the Xenium device e.g., transcriptomic data
  • is aligned with the data obtained from a method of the present disclosure e.g., images of the target nucleic acid).
  • a system of the present disclosure can comprise one or more nucleic acid constructs described herein, e.g., two or more, three or more, four or more or five or more nucleic acid constructs.
  • a system of the present disclosure can comprise a composition comprising one or more nucleic acid constructs described herein.
  • a system of the present disclosure can comprise a vector comprising one or more nucleic acid constructs described herein.
  • a system of the present disclosure can comprise a nucleic acid construct that comprises at least two promoter sequences upstream of the target nucleic acid, e.g., gRNA nucleotide sequence.
  • the first promoter is a promoter for expressing the target nucleic acid in live cells and the second promoter is a promoter for expressing the target nucleic acids in fixed cells.
  • the first promoter is a U6 promoter and the second promoter is a T7 promoter.
  • a system of the present disclosure can further comprise reagents for preparing a sample (e.g., in one or more reservoirs or containers).
  • a system of the present disclosure can comprise reagents for performing a fixation process, a permeabilization process and/or a decrosslinking process.
  • reagents comprise a fixative (e.g., an aldehyde), a permeabilizing reagent (e.g., an alcohol) and/or a decrosslinking reagent (e.g., sodium chloride and/or sodium bicarbonate).
  • a system of the present disclosure can further comprise reagents (e.g., in one or more reservoirs or containers) for performing an in vitro transcription reaction, e.g, an in vitro transcription reaction.
  • a system of the present disclosure can further comprise reagents for performing a reverse transcription reaction.
  • a system of the present disclosure can further comprise reagents for performing an amplification reaction, e.g., an RCA reaction.
  • a system of the present disclosure can further comprise reagents for performing a sequencing by synthesis reaction.
  • a system of the present disclosure can comprise reagents comprising one more of the following: polymerases (e.g., phi29 polymerase and/or Taq polymerase), RNA polymerases (e.g., T7 RNA polymerase), reverse transcriptases, nucleoside triphosphates or NTP analogues, primers, padlock probes, cofactors, ligation reaction reagents, endonucleases, lysis reagents, dyes, markers, RNase inhibitors and labels.
  • polymerases e.g., phi29 polymerase and/or Taq polymerase
  • RNA polymerases e.g., T7 RNA polymerase
  • reverse transcriptases e.g., reverse transcriptases
  • nucleoside triphosphates or NTP analogues e.g., primers, padlock probes, cofactors, ligation reaction reagents, endonucleases, lysis reagents
  • a system of the present disclosure can include one or more reducing agents, e.g., for performing an in vitro transcription reaction.
  • reducing agents include DTT (dithiothreitol), DTE (dithioerythritol), L- glutathione (GSH) and TCEP (Tris (2-Carboxyethyl) phosphine hydrochloride).
  • a system of the present disclosure includes DTT.
  • a system of the present disclosure can include DTT in a container at a concentration from about 1 mM to about 50 mM, e.g., about 5 mM.
  • a system of the present disclosure can comprise one or more detection probes, e.g., fluorescently labeled detection probes.
  • detection probes e.g., fluorescently labeled detection probes.
  • suitable containers comprise bottles, test tubes, vials and microtiter plates.
  • the containers can be formed from a variety of materials such as glass or plastic.
  • the system can comprise other materials or reagents desirable from a commercial and user standpoint, including other buffers and diluents.
  • a system of the present disclosure can include a buffer, e.g., a buffer for performing an in vitro transcription reaction, comprising one or more reducing agents, e.g, DTT.
  • the components of the system are provided in predetermined ratios, with the relative amounts of the various reagents suitably varied to obtain the desired sensitivity and throughput of the disclosed methods.
  • the present disclosure provides a method for imaging a target nucleic acid in a plurality of cells, comprising: a) providing a plurality of cells, wherein at least one cell of the plurality of cells comprises a nucleic acid construct comprising a first promoter, a second promoter and a target nucleic acid, wherein the first promoter and the second promoter are located upstream to the target nucleic acid in the nucleic acid construct; b) culturing the plurality of cells to allow expression of the target nucleic acid using the first promoter; c) fixing the plurality of cells to generate a plurality of fixed cells; d) transcribing the target nucleic acid in the at least one cell of the plurality of fixed cells using the second promoter; e) performing an amplification process to amplify the target nucleic acid; and f) imaging the amplified target nucleic acid in the at least one cell.
  • the target nucleic acid encodes a guide RNA (gRNA), a microRNA (miRNA), a small nucleolar RNA (snoRNA), a small interfering RNA (siRNA), piwi-interacting RNAs (piRNAs), aptamers, ribozymes, endogenous siRNAs (endo-siRNAs), a short hairpin RNA (shRNA) or a combination thereof.
  • gRNA guide RNA
  • miRNA microRNA
  • siRNA small nucleolar RNA
  • siRNA small interfering RNA
  • piRNAs piwi-interacting RNAs
  • aptamers ribozymes
  • endogenous siRNAs endo-siRNAs
  • shRNA short hairpin RNA
  • A3 The method of A2, wherein the target nucleic acid encodes a gRNA.
  • A4 The method of A3, wherein the gRNA has an editing efficiency greater than about 60%.
  • the present disclosure provides a method for performing a genomic screen, comprising: a) providing a plurality of cells, wherein at least one cell of the plurality of cells comprises a nucleic acid construct comprising a first promoter, a second promoter and a target nucleic acid encoding a gRNA, wherein the first promoter and the second promoter are located upstream to the target nucleic acid in the nucleic acid construct; b) culturing the plurality of cells to allow expression of the gRNA using the first promoter; c) fixing the plurality of cells to generate a plurality of fixed cells; d) transcribing the gRNA in the at least one cell of the plurality of fixed cells using the second promoter; e) performing an amplification process for amplifying the gRNA; and f) imaging the amplified gRNA in the at least one cell; and g) analyzing a change in a characteristic of the at least one cell of the plurality of cells associated with expression of the gRNA
  • B5. The method of B4, wherein the nucleotide sequence of the second promoter is incorporated into nucleotides 40 to about 200 located downstream from the 5’ end of the nucleotide sequence of the first promoter.
  • A-B7 The method of any one of A-B7, wherein the first promoter is selected from the group consisting of a U6 promoter, U3 promoter, U2 promoter, U5 promoter, Hl promoter, 7SK promoter, 75 J promoter, EF- la promoter, CMV promoter, a tRNA promoter, pGK promoter, SV40 promoter, CAG promoter, TRE promoter, VAI promoter and a combination thereof.
  • the first promoter is selected from the group consisting of a U6 promoter, U3 promoter, U2 promoter, U5 promoter, Hl promoter, 7SK promoter, 75 J promoter, EF- la promoter, CMV promoter, a tRNA promoter, pGK promoter, SV40 promoter, CAG promoter, TRE promoter, VAI promoter and a combination thereof.
  • B 11 The method of any one of A-B 10, wherein the second promoter is a promoter for a phage RNA polymerase.
  • B12 The method of Bl 1, wherein the second promoter is selected from the group consisting of a T3 promoter, a T7 promoter, a Sp6 promoter or a combination thereof.
  • B13 The method of Bl 2, wherein the second promoter is a T7 promoter.
  • B14 The method of the any one of A-B13, wherein the nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 1-18.
  • Bl 6 The method of Bl 5, wherein the rolling circle amplification process comprises: a) contacting the plurality of cells with (i) a padlock probe comprising two nucleotide sequences that are complementary to the target nucleic acid and (ii) a ligase to generate a circular DNA template; and b) performing a rolling circle amplification process to generate an amplicon from the circular DNA template.
  • Bl 7 The method of any one of A-B16, wherein the plurality of cells is permeabilized prior to performing the amplification process.
  • Bl 8. The method of any one of A-B17, wherein the plurality of cells is decrosslinked prior to performing the amplification process.
  • Bl 9 The method of any one of A-B18, wherein the fixing the plurality of cells to generate a plurality of fixed cells comprising contacting the plurality of cells with an aldehyde fixative.
  • nucleic acid construct further comprises a polynucleotide encoding a nuclease.
  • nucleic acid construct further comprises a barcode.
  • the plurality of cells further comprises a second cell comprising a second nucleic acid construct comprising the first promoter, the second promoter and a second target nucleic acid, wherein the first promoter and the second promoter are located upstream to the second target nucleic acid in the second nucleic acid construct.
  • B30 The method of any one of B-B29, wherein the plurality of cells further comprises a second cell comprising a second nucleic acid construct comprising the first promoter, the second promoter and a second target nucleic acid encoding a second gRNA, wherein the first promoter and the second promoter are located upstream to the second target nucleic acid in the second nucleic acid construct.
  • B32 The method of B or B31, wherein the characteristic is selected from the group consisting of cell viability, cell proliferation, cell size, cell morphology, cell motility, cell differentiation, cell adhesion, cell-cell contact, mutational status, karyotype, chromosomal aberrations, nucleic acid expression levels, nucleic acid localization, protein expression levels, protein localization, nucleic acid modifications, post-translational modifications, activation of a cell signaling pathway, repression of a cell signaling pathway, enzymatic activity, chromatin accessibility, histone modifications and other epigenetic changes, concentrations of cytokines and hormones, drug sensitivity, drug absorption and metabolism pharmacokinetics and pharmacodynamics, membrane potential and a combination thereof.
  • B34 The method of any one of A-B33, wherein the characteristic is nucleic acid expression levels.
  • B35 The method of any one of A-B34 further comprising performing an immunofluorescence process for detecting one or more target proteins.
  • the first promoter comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 4-5.
  • B37 The method of the any one of A-B36, wherein the second promoter comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 1-3.
  • nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 6-18.
  • nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 7-18.
  • nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 7.
  • nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 8.
  • nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 9.
  • nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 10.
  • nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 11.
  • nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 12.
  • nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 13.
  • nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 14.
  • nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 15.
  • nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 16.
  • nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 17.
  • nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 18.
  • the present disclosure provides a kit for performing the method of any one of A-B51.
  • the present disclosure provides a kit for performing a method for imaging a target nucleic acid in a plurality of cells, comprising: at least one container comprising the nucleic acid construct comprising a first promoter, a second promoter and a target nucleic acid, wherein the first promoter and the second promoter are located upstream to the target nucleic acid in the nucleic acid construct.
  • kits of D or DI wherein the nucleotide sequence of the second promoter is incorporated into nucleotides 40 to about 200 located downstream from the 5’ end of the nucleotide sequence of the first promoter.
  • D4 The kit of any one of D-D3, wherein the first promoter is a Pol III promoter or a Pol II promoter.
  • D5. The kit of any one of D-D4, wherein the first promoter is selected from the group consisting of a U6 promoter, U3 promoter, U2 promoter, U5 promoter, Hl promoter, 7SK promoter, 75 J promoter, EF-la promoter, CMV promoter, a tRNA promoter, pGK promoter, SV40 promoter, CAG promoter, TRE promoter, VAI promoter and a combination thereof.
  • kits of any one of D-D6, wherein the second promoter is a promoter for expression in fixed cells are provided.
  • D8 The kit of any one of D-D7, wherein the second promoter is a promoter for a phage RNA polymerase.
  • kits of D8, wherein the phage promoter is selected from the group consisting of a T3 promoter, a T7 promoter, a Sp6 promoter or a combination thereof.
  • kits of D9, wherein the phage promoter is a T7 promoter.
  • Dl l The kit of any one of D-D2, wherein the nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 1-18.
  • D12 The kit of any one of D-D2, wherein the target nucleic acid comprises from about 4 to about 1,000 nucleotides.
  • D13 The kit of any one of D-D2, wherein the target nucleic acid encodes a guide RNA (gRNA), a microRNA (miRNA), a small nucleolar RNA (snoRNA), a small interfering RNA (siRNA), piwi-interacting RNAs (piRNAs), aptamers, ribozymes, endogenous siRNAs (endo-siRNAs), a short hairpin RNA (shRNA) or a combination thereof.
  • gRNA guide RNA
  • miRNA microRNA
  • siRNA small nucleolar RNA
  • siRNA small interfering RNA
  • piRNAs piwi-interacting RNAs
  • aptamers ribozymes
  • endogenous siRNAs endo-siRNAs
  • shRNA short hairpin RNA
  • D14 The kit of D13, wherein the target nucleic acid encodes a gRNA.
  • DI 5 The kit of DI 4, wherein the gRNA has an editing efficiency greater than about 60%.
  • DI 6 The kit of any one of D-D 15, wherein the nucleic acid construct further comprises a polynucleotide encoding a nuclease.
  • DI 7 The kit of any one of D-D 16, wherein the nucleic acid construct further comprises a barcode.
  • D18 The kit of D17, wherein the barcode is located downstream of the target nucleic acid. DI 9. The kit of any one of D-D 18 further comprising dithiothreitol (DTT).
  • D20 The kit of any one of D-D 19, wherein the first promoter comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 4-5.
  • kits of any one of D-D22, wherein the nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 7-18.
  • the present disclosure provides a nucleic acid construct comprising a first promoter comprising a first nucleotide sequence, a second promoter comprising a second nucleotide sequence and a target nucleic acid, wherein the nucleotide sequence of the second promoter is incorporated into the nucleotide sequence of the first promoter.
  • E2 The nucleic acid construct of E or El, wherein the nucleotide sequence of the second promoter is incorporated into nucleotides 40 to about 200 located downstream from the 5’ end of the nucleotide sequence of the first promoter.
  • E3 The nucleic acid construct of E2, wherein the nucleotide sequence of the second promoter is incorporated into nucleotides 100 to about 200 located downstream from the 5’ end of the nucleotide sequence of the first promoter.
  • E4. The nucleic acid construct of any one of E-E3, wherein the first promoter is a promoter for expression in live cells.
  • E5. The nucleic acid construct of any one of E-E4, wherein the first promoter is a Pol III promoter or a Pol II promoter.
  • E6 The nucleic acid construct of any one of E-E5, wherein the first promoter is selected from the group consisting of a U6 promoter, U3 promoter, U2 promoter, U5 promoter, Hl promoter, 7SK promoter, 75J promoter, EF-la promoter, CMV promoter, a tRNA promoter, pGK promoter, SV40 promoter, CAG promoter, TRE promoter, VAI promoter and a combination thereof.
  • the first promoter is selected from the group consisting of a U6 promoter, U3 promoter, U2 promoter, U5 promoter, Hl promoter, 7SK promoter, 75J promoter, EF-la promoter, CMV promoter, a tRNA promoter, pGK promoter, SV40 promoter, CAG promoter, TRE promoter, VAI promoter and a combination thereof.
  • E7 The nucleic acid construct of E6, wherein the first promoter is a U6 promoter.
  • E8 The nucleic acid construct of E-E7, wherein the second promoter is a promoter for expression in fixed cells.
  • E9 The nucleic acid construct of E-E8, wherein the second promoter is a promoter for a phage RNA polymerase.
  • E10 The nucleic acid construct of E9, wherein the phage promoter is selected from the group consisting of a T3 promoter, a T7 promoter, a Sp6 promoter or a combination thereof.
  • El l The nucleic acid construct of E10, wherein the phage promoter is a T7 promoter.
  • nucleic acid of the any one of E-El 1 wherein the nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 1-18.
  • E13 The nucleic acid construct of any one of E-E12, wherein the target nucleic acid comprises from about 4 to about 1,000 nucleotides.
  • E14 The nucleic acid construct of any one of E-E13, wherein the target nucleic acid encodes a guide RNA (gRNA), a microRNA (miRNA), a small nucleolar RNA (snoRNA), a small interfering RNA (siRNA), piwi-interacting RNAs (piRNAs), aptamers, ribozymes, endogenous siRNAs (endo-siRNAs), a short hairpin RNA (shRNA) or a combination thereof.
  • gRNA guide RNA
  • miRNA microRNA
  • siRNA small nucleolar RNA
  • siRNA small interfering RNA
  • piRNAs piwi-interacting RNAs
  • aptamers ribozymes
  • endogenous siRNAs endo-siRNAs
  • shRNA short hairpin RNA
  • E15 The nucleic acid construct of E14, wherein the target nucleic acid encodes a gRNA.
  • E17 The nucleic acid construct of any one of E-E16, wherein the nucleic acid construct further comprises a polynucleotide encoding a nuclease.
  • E18 The nucleic acid construct of any one of E-E17, wherein the nucleic acid construct further comprises a barcode.
  • E19 The nucleic acid construct of El 8, wherein the barcode is located downstream of the target nucleic acid.
  • E20 The nucleic acid construct of any one of E-E19, wherein the nucleotide sequence of the first promoter comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 4-5.
  • E21 The nucleic acid construct of any one of E-E20, wherein the nucleotide sequence of the second promoter comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 1-3.
  • E22 The nucleic acid construct of any one of E-E21, wherein the nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 6-18.
  • E23 The nucleic acid construct of any one of E-E22, wherein the nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 7-18.
  • the present disclosure provides a nucleic acid comprising a first promoter comprising a first nucleotide sequence, a second promoter comprising a second nucleotide sequence, wherein the nucleotide sequence of the second promoter is incorporated into the nucleotide sequence of the first promoter, wherein the nucleic acid comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 7-18.
  • F 1 The nucleic acid construct of F, wherein the nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 7.
  • nucleic acid construct of F wherein the nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 8.
  • nucleic acid construct of F wherein the nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 9.
  • nucleic acid construct of F wherein the nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 10.
  • nucleic acid construct of F wherein the nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 11.
  • nucleic acid construct of F wherein the nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 12.
  • nucleic acid construct of F wherein the nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 13.
  • nucleic acid construct of F wherein the nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 14.
  • nucleic acid construct of F wherein the nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 15.
  • nucleic acid construct of F wherein the nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 16.
  • nucleic acid construct of F wherein the nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 17.
  • nucleic acid construct of F wherein the nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of SEQ ID NO: 18.
  • F13 The nucleic acid construct of F, wherein the nucleic acid comprises the nucleotide sequence of any one of SEQ ID NOs: 7-18.
  • G The present disclosure provides a composition comprising the nucleic acid construct of any one of E-F13.
  • the present disclosure provides a method for imaging a target nucleic acid in a plurality of cells, comprising: a) providing a plurality of cells, wherein at least one cell of the plurality of cells comprises a nucleic acid construct comprising (i) a target nucleic acid and (ii) a hybrid promoter located upstream to the target nucleic acid in the nucleic acid construct, wherein the hybrid promoter comprises a first promoter and a second promoter, and wherein the nucleotide sequence of the second promoter is incorporated into the nucleotide sequence of the first promoter; b) culturing the plurality of cells to allow expression of the target nucleic acid using the first promoter of the hybrid promoter; c) fixing the plurality of cells to generate a plurality of fixed cells; d) transcribing the target nucleic acid in the at least one cell of the plurality of fixed cells using the second promoter of the hybrid promoter; e) performing an amplification process to amplify the hybrid
  • the present disclosure provides a method for imaging a target nucleic acid in a plurality of cells, comprising: a) providing a plurality of cells, wherein at least one cell of the plurality of cells comprises a nucleic acid construct comprising a first promoter, a second promoter and a target nucleic acid, and wherein the first promoter and the second promoter are located upstream to the target nucleic acid in the nucleic acid construct; b) culturing the plurality of cells to allow expression of the target nucleic acid using the first promoter; c) fixing the plurality of cells in a fixative comprising aldehyde to generate a plurality of fixed cells; d) decrosslinking the plurality of fixed cells to generate a plurality of decrosslinked cells; e) transcribing the target nucleic acid in at least one cell of the plurality of decrosslinked cells using the second promoter of the hybrid promoter; f) performing an amplification process to amplify the target nucleic acid
  • aldehyde is formaldehyde, paraformaldehyde, glutaraldehyde or a combination thereof.
  • nucleotide sequence of the first promoter comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 4-5.
  • nucleotide sequence of the second promoter comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 1-3.
  • nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 1-18.
  • nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 6-18. 19.
  • nucleic acid construct comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 7-18.
  • imaging the amplified target nucleic acid comprises imaging the target nucleic acid by in situ sequencing.
  • any one of H-I10 further comprising analyzing a change in a characteristic of one or more cells in the plurality of cells expressing the target nucleic acid compared to one or more cells in the plurality of cells that does not express the target nucleic acid.
  • the method of Il l wherein the characteristic is selected from the group consisting of cell viability, cell proliferation, cell size, cell morphology, cell motility, cell differentiation, cell adhesion, cell-cell contact, mutational status, karyotype, chromosomal aberrations, nucleic acid expression levels, nucleic acid localization, protein expression levels, protein localization, nucleic acid modifications, post-translational modifications, activation of a cell signaling pathway, repression of a cell signaling pathway, enzymatic activity, chromatin accessibility, histone modifications and other epigenetic changes, concentrations of cytokines and hormones, drug sensitivity, drug absorption and metabolism pharmacokinetics and pharmacodynamics, membrane potential and a combination thereof.
  • the characteristic is selected from the group consisting of cell viability, cell proliferation, cell size, cell morphology, cell motility, cell differentiation, cell adhesion, cell-cell contact, mutational status, karyotype, chromosomal aberrations, nucleic acid expression
  • the present disclosure provides a method for analyzing one or more characteristics of a plurality of cells, comprising: a) providing a plurality of cells, wherein at least one cell of the plurality of cells comprises a nucleic acid construct comprising a first promoter, a second promoter and a target nucleic acid, and wherein the first promoter and the second promoter are located upstream to the target nucleic acid in the nucleic acid construct; b) culturing the plurality of cells to allow expression of the target nucleic acid using the first promoter of the hybrid promoter; c) fixing the plurality of cells to generate a plurality of fixed cells; d) detecting one or more characteristics of the at least one cell of the plurality of fixed cells; e) transcribing the target nucleic acid in the at least one cell of the plurality of fixed cells using the second promoter of the hybrid promoter; f) performing an amplification process to amplify the target nucleic acid; g) imaging the amplified target nucle
  • JI The method of J, wherein the characteristic is selected from the group consisting of cell viability, cell proliferation, cell size, cell morphology, cell motility, cell differentiation, cell adhesion, cell-cell contact, mutational status, karyotype, chromosomal aberrations, nucleic acid expression levels, nucleic acid localization, protein expression levels, protein localization, nucleic acid modifications, post-translational modifications, activation of a cell signaling pathway, repression of a cell signaling pathway, enzymatic activity, chromatin accessibility, histone modifications and other epigenetic changes, concentrations of cytokines and hormones, drug sensitivity, drug absorption and metabolism pharmacokinetics and pharmacodynamics, membrane potential and a combination thereof.
  • the characteristic is selected from the group consisting of cell viability, cell proliferation, cell size, cell morphology, cell motility, cell differentiation, cell adhesion, cell-cell contact, mutational status, karyotype, chromosomal aberrations, nucleic acid
  • J2 The method of J or JI further comprising decrosslinking the cells prior to transcribing the target nucleic acid.
  • J3 The method of J2, wherein detecting the characteristic of the at least one cell is performed after fixation and prior to decrosslinking.
  • J4 The method of any one of J-J3, wherein the characteristic is protein expression levels.
  • J5. The method of any one of J-J4, wherein the characteristic is nucleic acid expression levels.
  • J6 The method of any one of J-J5, wherein the nucleotide sequence of the second promoter is incorporated into the nucleotide sequence of the first promoter.
  • J6.1 The method of any one of J-J5, wherein the nucleotide sequence of the second promoter is incorporated into nucleotides 40 to about 200 located downstream from the 5’ end of the nucleotide sequence of the first promoter.
  • J7 The method of any one of J-J6.1, wherein the nucleotide sequence of the second promoter is incorporated into nucleotides 100 to about 200 located downstream from the 5’ end of the nucleotide sequence of the first promoter.
  • J8 The method of any one of J-J7, wherein the nucleotide sequence of the first promoter comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 4-5.
  • nucleotide sequence of the second promoter comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 1-3.
  • nucleic acid of the hybrid promoter comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 1-18.
  • JI 1 The method of any one of J-J10, wherein the nucleic acid of the hybrid promoter comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 6-18. J12.
  • nucleic acid of the hybrid promoter comprises a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 7-18.
  • the present disclosure further provides a system for performing the method of any one of A-B51 and H-J12.
  • the present disclosure further provides a system comprising the nucleic acid construct of any one of E-F13.
  • HEK 293T, 3T3, L929, Raw 264.7, DLD-1 and A549 cells were all obtained from ATCC and maintained in DMEM supplemented with 10% Fetal Bovine Serum (FBS; v/v), 100 units/ml penicillin, 100 pg/ml streptomycin and IX GlutaMAX.
  • FBS Fetal Bovine Serum
  • IMR-90 fibroblasts were obtained from ATCC (ATCC CCL-186) and maintained in EMEM (ATCC 30-2003) supplemented with 10% (v/v) FBS.
  • iPSCs For iPSC-derived neurons, iPSCs (ALSTEM, iPHN) were differentiated using a strategy combining NGN2 programming and small molecule patterning optimized from a previously published protocol (Nehme et al., Cell Rep. 23, 2509-2523 (2016)). On day 7 of differentiation, cells were lifted from the flask and plated at 40,000 cells per well in poly-d- lysine and iMatrix-coated 96-well plates. On day 8, cells were transduced by adding appropriate volumes of lentiviral supernatant overnight to achieve an MOI of ⁇ 0.3. Cells were then subjected to antibiotic selection by puromycin at 1 pg/ml on day 11 for 3 days.
  • iAstrocytes were generated from iPSCs (ALSTEM, iPl l) based on a published protocol (Chailangkam et al., Nature 536, 338-343 (2016)). Immature astrocytes harvested from astrospheres were plated at 10,000 cells per well in matrigel coated 96-well plates for further maturation. A day later, cells were transduced by adding appropriate volumes of lentiviral supernatant overnight to achieve an MOI of ⁇ 0.3. Cells were selected by puromycin treatment at 2 pg/ml at day 5 of maturation for 3 days and fixed at day 8 with 4% paraformaldehyde in IX PBS for 30 minutes.
  • Immortalized macrophages (ER-Hoxb8-immortalized murine myeloid progenitor cells (Luchetti, G. et al., Cell Host Microbe 29, 1521-1530. elO (2021); Chen et al., Mol Ther Methods Clin Dev 27, 431-449 (2022)) were established from Cas9 transgenic mice (Platt et al., Cell 159, 440-455 (2014)). Cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS, 20 ng/mL murine granulocyte-macrophage colonystimulating factor (GM-CSF, eBioscience), and 1 mM b-estradiol (Millipore Sigma).
  • GM-CSF murine granulocyte-macrophage colonystimulating factor
  • Cells were transduced by spinfection at 3200 x g at 32°C for 45 min with 5 pg/ml polybrene, cultured for another 2 days and then selected with 10 pg/ml puromycin. Cells were differentiated into macrophages in DMEM supplemented with 10% (v/v) FBS, and 20% (v/v) L929-conditioned medium at 37°C with 5% CO2 and were re-plated to 96-well plates at 20,000 cells per well on day 5 for experiments.
  • Bone marrow was harvested from tibias and femurs of 12-18 weeks old Cas9-eGFP transgenic mice (Platt et al. (2014)). After red blood cells lysis (ACK lysis buffer, GIBCO A1049201), 20 M cells were plated in BMDM media [DMEM high glucose, 10% Tet- Negative heat-inactivated FBS, IX GlutaMAX, lOOU/ml penicillin-streptomycin and lOOng/ml recombinant murine M-CSF (Genentech media facility)] at a density of 0.8M cells/ml in 150-mm non-TC treated dishes (Corning). Cells were transduced on day 3 by adding a single lentivirus or lentiviral pool supernatants overnight in fresh BMDM media. Transduced cells were selected with 5 pg/ml puromycin from day 8 to day 11.
  • BMDM media DMEM high glucose, 10% Tet- Negative heat-inactivated F
  • T cells were isolated using the pan-T cell isolation kit (Miltenyi) and frozen in FBS supplemented with 10% DMSO. Following thawing, T cells were resuspended in T cell media (X VIVO 15 + 5% FBS + 500U/mL IL-2) to a concentration of IxlO 6 cells/mL, and stimulated with anti-human CD3/CD28 CTS Dynabeads (ThermoFisher Scientific, catalog no. 40203D) at a 1 : 1 celkbead ratio. Approximately 18 hours post-stimulation, cells were transduced with Lenti-Cas9 (2.5% v/v).
  • sgRNA constructs contained a puromycin selection cassette, cells were then selected with 2pg/mL puromycin on day 3. Over the next few days, cells were expanded and maintained by adding media or splitting as necessary. On day 6, CRISPR-mediated Target 1 knockout was assessed by flow cytometry. Cells were then plated onto CD3 antibody coated lysine surfaces, allowed to adhere for 15 minutes at 37°C before 4% PFA fixation followed by ISS.
  • All cells were cultured at 37°C and 5% CO2 in a humidified incubator.
  • mice All mice were maintained in a pathogen-free animal facility under standard animal room conditions (temperature 21 ⁇ 1°C; humidity 55%-60%; 12h light/dark cycle).
  • Imaging was performed on a Nikon Ti2 microscope equipped with Nikon lenses, a Hammamatsu Fusion BT camera and a Lumencor CELESTA light engine.
  • In situ sequencing cycles were imaged using either a 10X 0.45 NA CFI Plan Apo Lambda or a 4X 0.20 NA CFI Plan Apo Lambda objective with the CELESTA- DA/FETR/Cy5/Cy7 (Semrock) excitation filter and FF01-391/477/549/639/741 dichroic unless noted otherwise.
  • Laser excitation wavelength and emission filter are set up for each channel: DAPI (408nm, FFO 1-441/511/593/684/817); base G (545nm, FFO 1-575/19); base T (545nm, FF01-615/24); base A (637nm, FF01-680/42); base C (637nm, FF01-732/68).
  • DAPI 365nm, FF01-433/2
  • CF430 446nm, 5%, ex: FF01- 432/523/702, em: FF01-470/28, dm: FF459/526/596-Di01).
  • HCR FISH phenotypic images were acquired with a 20X 0.75NA CFI Plan Apo Lambda objective with the following conditions: DAPI (365nm, FF01- 441/511/593/684/817); AF594 (561nm, FF01-615/24); AF647 (637 nm, FF01-680/42), AF750 (748nm, FFO 1-792/64).
  • IBEX Immunofluorescence images were acquired with a 20X 0.75NA CFI Plan Apo Lambda objective the following conditions: DAPI (365nm, FF01-441/511/593/684/817); CF430 (446nm, ex: 432/523/702, em: FF01-470/28, dm: 459/526/596); GFP (477nm, FF01- 511/20); AF532 (545nm, ex: FFO 1-432/523/702, em: FF01-563/9, dm: 555-Di03); AF647 (637nm, FF01-680/42); AF750 (748nm, em: FF01-792/64).
  • DAPI 200 ms
  • base G 200 ms
  • base C 200 ms
  • base A 200 ms
  • base T 800 ms
  • 10% and 4% overlapping regions were acquired for lOx and 4x objective lenses, respectively, to use the same physical distance for the image registration.
  • the same exposure was also used for the frameshift reporter assay at 4x magnification.
  • HEK293T cells were seeded into 6-well plates at a density of 1 million cells per well. The following day, cells were transfected with the expression plasmid, delta8.9 and pCMV-VSV-G at a molar ratio of (1 :2.3:0.2) using Lipofectamine 3000 (ThermoFisher Scientific L3000015). Opti-MEM (Gibco 31985062) was exchanged to culture media after 4 hours. Viral supernatant was harvested 2 days after transfection and filtered through a 0.45 pm filter. For infection of BMDMs, viral supernatant was further concentrated by adding Lenti-X concentrator (Takara 31231) and centrifuging at 4°C for Jackpot. LentivirusL titer was quantified before the screens using CellTiter-Glo kit (Promega G7570).
  • Cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences 15714) in PBS for 30 min, and washed three times with PBS with 0.05% Tween (PBST). Cells were permeabilized with 70% ethanol for 30 minutes, followed by 75% volume exchange with PBST three times to prevent drying and two complete PBST washes. Afterwards, heatdecrosslinking was carried out in 0. IM sodium bicarbonate and 0.3M NaCl in water at 65°C for 4 hours (cell culture) to 24 hours (tissue). Glass-bottom plates that tolerate high heat can be used (Cellviz, P96-1.5H-N or P12-1.5H-N).
  • IVT was conducted using the HiScribe® T7 Quick High Yield RNA Synthesis Kit (NEB E2050S) - lx T7 RNA Polymerase Mix, NTP Buffer Mix at 10 mM with the addition of RiboLock RNase inhibitor (ThermoFisher Scientific EO0384) at 0.4U/pl at 37 °C for 12 h.
  • RiboLock RNase inhibitor ThermoFisher Scientific EO0384
  • FIGS. 9A- 9C which uses the original OPS protocol (Feldman et al., Cell 179, 787-799. el7 (2019)).
  • An amine-modified primer was hybridized at 1 pM in PBST for 30 min at room temperature. After the hybridization and before RT, cells were fixed with 3.2% paraformaldehyde and 0.1% glutaraldehyde (Electron Microscopy Sciences 16120) in PBST for 30 min.
  • the reaction was quenched by adding 0.2M Tris-HCl (pH 8), and cells were washed three times with PBST.
  • the reverse transcription reaction mix (lx RevertAid RT buffer, 250 pM dNTPs (NEB N0447L), 0.2 mg/mL recombinant albumin (NEB B9200S), 1 pM RT primer, 0.4 U/pL Ribolock RNase inhibitor, and 4.8 U/pL RevertAid H minus reverse transcriptase (ThermoFisher Scientific EP0452) was added into samples, followed by incubation at 37 °C overnight.
  • cells were incubated in gap-fill reaction mix (lx Ampligase buffer, 0.4 U/pL RNase H (Enzymatics Y9220L), 0.2 mg/mL recombinant albumin, 100 nM padlock probe, 0.02 U/pL TaqIT polymerase (Enzymatics P7620L), 0.5 U/pL Ampligase (Lucigen A3210K) and 50 nM dNTPs) for 5 min at 37 °C and 90 min at 45 °C.
  • gap-fill reaction mix lx Ampligase buffer, 0.4 U/pL RNase H (Enzymatics Y9220L), 0.2 mg/mL recombinant albumin, 100 nM padlock probe, 0.02 U/pL TaqIT polymerase (Enzymatics P7620L), 0.5 U/pL Ampligase (Lucigen A3210K) and 50 nM dNTPs
  • a targeting sgRNA was transduced by lentivirus to Cas9-expressing cells.
  • cells were cultured for 4 days and then fixed in 4% paraformaldehyde in PBS for 30 min. Cells were washed twice with PBS and then incubated with fluorophore-conjugated antibodies (Target 1 antibody or Target 2 antibody) for Ih followed by a PBS wash.
  • Fluorophore-conjugated antibodies Target 1 antibody or Target 2 antibody
  • Antibody signal was measured by flow cytometry (Sony, MA900 Multi-Application Cell Sorter) gated on live cells based on forward and side scatter.
  • Cas9-expressing A549 cells were first transduced with the frameshift reporter (Kudo et al., Cell Syst 13, 376-387. e8 (2022)) at high MOI by repeating the transduction processes and selected with 300 pg/ml hygromycin (ThermoFisher Scientific 10687010) for 1 week.
  • Five targeting and five non-targeting control sgRNAs were individually cloned into a PerturbView vector, pooled equimolarly, packaged as lentivirus, and transduced into Cas9- expressing A549 cells at low MOI, followed by puromycin selection.
  • Cells were plated 12,000 cells per well in a 96-well plate (CellViz, P96-1.5H-N) and fixed with 4% paraformaldehyde for 30 min on the following day. Epitope staining was conducted as previously described (Feldman et al. (2019)), followed by in situ sequencing. As described above, 4-fold longer durations of exposure were used for standard OPS than for PerturbView, due to the different dynamic ranges of the two methods.
  • cells were fixed with 4% PFA for 30 min or with a 3: 1 mix of methanol and acetic acid for 20 min.
  • cells were incubated in the decrosslinking buffer either at room temperature (25°C) or 65°C for 4 hours.
  • sgRNAs were designed per gene for 23 NFicB-related genes, 130 metabolic and signaling genes (Table 2) and 10 olfactory receptor genes (as nonexpressed gene controls), along with 60 different non-targeting sgRNAs. Spacer sequences were generated by crisprVerse to design barcodes that can be uniquely identified by 6 and 8 cycles of in situ sequencing for NTC and BMDM libraries, respectively.
  • the oligo pool was synthesized by Twist Bioscience, amplified by dial-out PCR using Ex Premier DNA Polymerase (Takara Bio), cloned into a Perturb View vector with golden gate cloning using BsmBI, transformed into Stellar electrocompetent cells (Takara Bio), and packaged into a lentivirus library while maintaining >1000x coverage at every step.
  • gDNA was extracted and amplified according to an established protocol (Feldman et al. (2022). Indexed amplicons were sequenced on an Illumina MiSeq with 150 cycles and the v3 reagent kit (Illumina, MS-102-3001). The reads were aligned to the expected amplicon sequences (N20 corresponding to the spacer region flanked by backbone sequences) using the Burrows-Wheeler alignment tool (bwa-mem) to produce reads counts for each designed guide.
  • bwa-mem Burrows-Wheeler alignment tool
  • BMDMs were isolated from mice expressing Cas9-EGFP and cultured in DMEM high glucose, 10% FBS, 2 mM glutamine, 1% Pen/Strep, and 100 ng/ml CSF-1. After two days, these cells were split into new plates and infected with the guide-bearing lentiviruses at MOI of ⁇ 0.2. Media was replaced the following day and selection of the infected BMDMs began with 5 pg/ml puromycin. Three days after the selection and media change, cells were plated in a 12-well plate (Cellviz P12-1.5H-N) at a density of 276,000 cells per well.
  • cells were stimulated with 10 ng/ml TNFa, 2 ng/ml ILip, 100 ng/ml LPS and H2O for 40 min before fixation.
  • Cells were fixed in 4% paraformaldehyde in PBS for 30 min.
  • cells were permeabilized with 0.2% Triton-X in PBS for 10 min.
  • Cells were then blocked with 3% BSA in PBS for 45 min, incubated with p65 antibody in PBS at 1 : 1000 (Cell Signaling Technologies #8242) for 1 h and washed three times with PBST.
  • BMDMs were transduced, selected and plated in the same manner as above.
  • Cells were stimulated with 10 ng/ml TNFa, 2 ng/ml ILip for 24 h.
  • Cells were fixed in 4% paraformaldehyde in PBS for 30 min, washed three times with PBST and then permeabilized with 0.2% Triton-X for 10 min, followed by three washes with PBST.
  • HCR FISH HCR FISH
  • probe hybridization buffer (30% formamide, 5x SSC, 0.1% Tween)
  • primary HCR FISH probes (Molecular Instruments) against Gbp2 (B2, Table 1C), Ccl4 (B3, Table 1C) and Ripk2 (B5, Molecular Instruments) diluted at 1 :250 ratio in probe hybridization buffer with 0.4 U/pL Ribolock at 37 °C for 4 hours.
  • Samples were then washed with HCR probe hybridization buffer four times, each for 5 min at 37 °C.
  • samples were incubated in HCR amplification buffer (5x SSC, 0.1% Tween) at room temperature for 30 minutes.
  • HCR amplifiers were separately heated at 95 °C for 90s and then cooled to room temperature in the dark for 30 min.
  • HCR amplifier mix (Molecular Instruments, Bl-488 hl/h2, B2-594 hl/h2, B3-647 hl/h2 diluted at 1 : 125 in amplification buffer with 0.4 U/pL Ribolock) were added to the sample, followed by incubation at room temperature for 2 hours. Excess amplifiers were removed by washing five times with HCR probe amplification buffer. An imaging buffer (200 ng/mL DAPI in 2X SSC with) with 0.4 U/pL Ribolock was then added to the sample, followed by a 10 min incubation.
  • HCR FISH probes were stripped off by adding HCR stripping solution (80% formamide in 2X SSC) and incubated at 37°C for 30 min, followed by three washes at 37°C with HCR stripping buffer, each for 5 min, followed by three PBST washes.
  • HCR stripping solution 80% formamide in 2X SSC
  • BMDMs were transduced with the NTC library at an MOI below 20%, selected and plated in the same manner as above.
  • Cells were fixed in 4% paraformaldehyde in PBS for 30 min, washed three times with PBST. Control wells were kept in PBST with 0.4 U/pL Ribolock.
  • cells were permeabilized with 0.2% Triton-X in PBS for 10 min, washed three times with PBST, and blocked with 3% BSA in PBS for 1 h at room temperature.
  • Cells were stained with an antibody against F4/80 (1 : 100 in PBS with 0.4 U/pL Ribolock, #27076S Cell Signaling Technology) for 45 min, followed by three washes with PBST. Immunofluorescence was repeated for 3 or 6 cycles using either 4i40, IBEX27 and cycIF45 following published protocols. After immunofluorescence, the PerturbView protocol was resumed as written above at the ethanol permeabilization step.
  • DLD-1 cells were transfected with the NTC library with polybrene at MOI ⁇ 0.2. Transfected cells were selected and maintained with 2 pg/ml puromycin. 1.5 million transfected cells were subcutaneously implanted into each 8-12 weeks old female NCR nude mouse (Taconic).
  • mice were anesthetized for manual restraint with isoflurane gas to facilitate injection and minimize stress to the animal. As soon as mice were immobile, they were removed from the chamber and placed on a nose cone delivering the anesthetic gas. The injection site area was cleaned with alcohol. Using forceps, the skin over the inoculation site was lifted and a sterile 25G needle attached to a 1.0ml tuberculin syringe was inserted through the skin into the subcutaneous space. An inoculum dose of at most 200 pl was deposited. The needle was then held in place for approximately 3 seconds before being carefully withdrawn. The mouse was returned to its cage and monitored until fully recovered from anesthesia.
  • DLD-1 cells developed into subcutaneous tumors of ⁇ 250mm. Individual xenografts were harvested and halved, with half the tumor chunk embedded in OCT and frozen on dry ice immediately and the other half fixed in 10% neutral buffered formalin for 20 hours at 4°C, processed and embedded in paraffin. Fresh frozen and FFPE tumor blocks were cut into 10pm and 5pm thick tissue sections, respectively, placed on Superfrost Plus Microscope glass slides or Xenium slides, and stored at -80°C for fresh frozen tissue and 4°C for FFPE tissue.
  • Fresh frozen samples were taken from -80°C and fixed with 4% paraformaldehyde for 30 min in a 50 ml falcon tube. Slides were washed three times by transfer to new 50mL tubes filled with PBS for 5 min each. Each slide was transferred to a tube filled with 200 ng/ml DAPI in PBS and mounted on a SecureSeal hybridization chamber to acquire nuclear images. After imaging, slides were transferred to a tube filled with decrosslinking buffer (0. IM sodium bicarbonate and 0.3M NaCl in water) and incubated at 65°C overnight. After PBS washes, slides were mounted on Xenium cassettes (lOx Genomics, PN-1000566).
  • Downstream reactions including IVT, RT, gap-filling and RCA, took place in the Xenium cassettes until the in situ sequencing step. Then, a SecureSeal Hybridization chamber was mounted for in situ sequencing and imaging. Z-stack images were acquired -8 to 20 pm from the tissue plane with 4 pm steps.
  • FFPE sections were first deparaffinized and de-crosslinked according to the Xenium FFPE protocol (Janesick et al. bioRxiv (2022) doi: 10.1101/2022.10.06.510405). Briefly, FFPE sections were incubated at 60°C for 2 hours, equilibrated to room temperature for 7 minutes, and immersed in xylene and incubated for 10 mins twice. FFPE sections were then immersed in an ethanol series of 100%, 100%, 96%, 96% and 70% for 3 minutes for each incubation. Finally, sections were rinsed in nuclease-free water for 20 seconds and assembled into Xenium cassettes.
  • Each section was decrosslinked with 300 pl of Xenium decrosslinking buffer and incubated at 80°C for 30 minutes, 22°C for 10 minutes, and washed with PBST for 1 minute twice before acquiring DAPI images.
  • Perturb View IVT and ISS protocols were performed as described herein.
  • PRKDC Perturb View protocol
  • phenotypic images were acquired using 20x magnification for resolution and in situ sequencing was acquired at lOx magnification for speed. Due to a jitter introduced between imaging cycles and the chromatic aberrations between objective lenses, a series of alignments was performed.
  • images were stitched together using ASHLAR to represent a whole-well image.
  • Composite well images were then resized 2x by nearest-neighbor interpolation to match the pixel size of phenotyping images. Images across cycles were then cropped to the same size (minimum image size obtained from each well across cycles). These whole-well images across cycles were then coarsely aligned by ASHLAR and split into smaller tiles of 5000 x 5000 pixels. Affine transformation followed by optical flow-based registration was performed using microaligner for further linear and non-linear alignment.
  • nuclei were segmented using Stardist, and the resulting nuclei served as seeds for cytoplasmic segmentation using watershed. Segmentation information was stored in Zarr format for use along the pipeline. Spot detection in cell culture:
  • Reads were identified by calling the base with the highest intensity at each spot identified in the first step, utilizing an ad-hoc quality score per sequencing call, based on the highest and second-highest intensity channels, calculated as:
  • a table containing barcode information and associated quality scores was then used to assign the most common barcode reads to each cell.
  • the resulting table includes all cells with sequencing reads, listing the top two most common barcode sequences for each cell.
  • Intensity features such as percentiles (0.25, 0.75), median absolute deviation (MAD), maximum, median and mean intensity, intensity standard deviation, and total intensity (sum), were calculated using the region_props module of scikit-image package v0.20.0.
  • Other features included peak intensity and locations, number of peaks, intensity distribution (mean intensity and radial coefficient of variation for each concentric bin), edge intensity (min, mean, max, sum, std along edge pixels), weighted center of mass and mass displacement, and the Hu moments (translation, scale and rotation invariant) of the intensity image.
  • Results from in situ sequencing were merged with phenotypic imaging data based on their common cell label.
  • the resulting table contained all the features listed above, for each channel of the phenotype image, along with the label of each cell, well identity, barcodes identified, and barcode associated information (ID, gene, chromosome, guide name, etc.).
  • the alignment was evaluated by checking the overlap between the segmentation mask and DAPI channel image from each cycle.
  • a rectangular bounding box was defined for each cell based on the segmented nuclei.
  • the pixel-wise correlation inside the bounding box between the first cycle and each later cycle was calculated. The correlation is close to 1 if the DAPI image and nuclear mask overlaps, indicating little to no alignment error.
  • Cells were removed from analysis if the minimum correlation coefficient across cycles were less than 0.9. Cells were further filtered out by removing small nuclei (area ⁇ 400 pixels).
  • a method was developed to compute distinct thresholds, by adopting an approach used in HTODemux, where barcode assignment also requires a background estimate for each batch and cell hashing oligo.
  • a local maximum was computed within the corresponding cell labels (cell segmentation for standard OPS and nucleus segmentation for Perturb View) and those pixels were collected as potential reads (z.e., each pixel position contains a vector of length 4 corresponding to each channel). For efficiency, the number of pixels for the following computation was limited to 25,000.
  • the top 0.5 percentile of cells were excluded as outliers, and a negative bimodal distribution was fit to the obtained cluster.
  • the 99 th percentile of the fitted distribution was taken as the threshold for each channel. Each channel was thresholded accordingly and the remaining spots were segmented as reads.
  • MCF7 cells were transduced at high MOI. Thus, focus was placed on the spot intensities over the fraction of cells with spots. After the spots were identified as described above, maximum intensities of the Laplacian-of-gaussian-transformed images were computed for each spot and channel. The mean of the highest intensities across four channels were then computed for each field- of-view.
  • HA-positive and HA-negative To classify each cell into HA-positive and HA-negative, a two-component Gaussian mixture model was fitted to the distribution of mean HA intensity. A probability threshold of 0.95 was used to remove uncertain cells from data. Note that false positives (HA-negative cells with a targeting guide) could be attributed not only to barcode diffusion but other causes, such as reporter variability, multiple integrants and recombination or downstream analysis errors in segmentation and base calling.
  • Phenotypic features were extracted from two rounds of imaging as described above. DAPI replicates were excluded from later phenotyping rounds and Cas9-GFP signal was excluded from analysis. 131 features that contained > 2% missing values (NaN or Inf) were removed, and cells containing the remaining missing values were also discarded. Data were then normalized by a well-wise robust Z-scoring on the NTC population. Features with NTC IQR less than 0.1 were removed, followed by retaining only 356 features whose standard deviation lies within the range of 0.1 to 5.0. Outlier values with a normalized score >5 were discarded by removing any features with >0.1% outliers as well as any remaining cells with outlier values. Finally, guides with ⁇ 15 remaining cell counts were also discarded. PCA was applied to keep 95% of the variance, resulting in 55 principal components. The filtering retained 205,488 cells from 215,275 cells.
  • PCA features were subjected to sphering based on NTC population, aggregated across cells and re-centered on the mean of the negative controls. The centered features were further aggregated by taking the median across guides. Leiden clustering was performed to these features but without NTC with resolution ⁇ ! .0.
  • PCA features were transformed by robust Z-score normalization based on NTC population.
  • Nuclear segmentation was performed using Stardist and expanded 5 pixels by dilation to account for registration errors. Each tissue section contains mouse cells surrounding the tumors and a necrotic region that does not produce barcodes. To obtain relevant statistics from the transduced cells, segmentation masks corresponding to non- necrotic tumor tissue were chosen visually using Napari viewer. Each sample replicate contained at least 27,802 cells for analysis.
  • Immunofluorescence images were corrected and stitched as described above. Nucleus segmentation was performed using Stardist on DAPI channel. In situ sequencing images were also corrected and stitched as described above, and registered to the immunofluorescence images using WSIReg91 using a rigid transformation followed by a nonlinear spline transformation. Nuclei were dilated by 2 pixels to account for segmentation and registration errors.
  • a maximum filter with a kernel size of 9 pixels was applied to 4 channel images of the in situ sequencing. Separately, local maxima of the Laplacian of Gaussian images were computed and aggregated across channels for potential peaks. Peaks within a 5-pixel radius were aggregated by sampling the brightest intensity for each channel. Peaks less than an intensity threshold were filtered out. Peaks were then linked over cycles based on location using trackpy with a search radius of 10 pixels. Peaks that were present in at least 5 cycles were retained for further analysis, and the missing cycle was imputed if the other 5 cycles were uniquely mapped to a library. Cross-talk of channel intensities was corrected as described above. Bases corresponding to the maximum intensity across channels were assigned as a read sequence.
  • Top and bottom Shannon diversity quartiles were defined as high and low diversity, respectively. Differential analysis was performed between these two groups using Wilcoxon test. Genes qualified as hits if they had a log fold-change greater than 0.5, fold-change greater than 2.5 and, were expressed in at least 20% of the population, and ranked in the top 10 when sorted by scores.
  • This example describes Perturb View, an Optical pooled screen (OPS) technology that leverages in vitro transcription (IVT) to amplify barcodes prior to ISS, thereby enabling screens with highly multiplexed phenotypic readouts across diverse systems, including primary cells and tissues.
  • OPS Optical pooled screen
  • IVTT in vitro transcription
  • Optical pooled screens facilitate image-based phenotyping under thousands of perturbations. These screens, however, are currently limited to low-plex phenotyping and cancer cell lines, primarily due to the inefficiency of the current in situ sequencing technique. To overcome such inefficiencies, this example describes an exemplary method that utilizes in vitro transcription for detecting gRNA expression in a biological sample. The exemplary method described in this example also allows for extensive non-destructive phenotyping in combination with in situ sequencing.
  • FIG. 1A and FIG. 9A An exemplary method of the present disclosure is shown in FIG. 1A and FIG. 9A.
  • FIG. 2 the instability of in situ sequencing techniques in various cell types including neuron, macrophages, dendritic cells and microglia makes the integration of imaging techniques difficult.
  • A549 and iPSC-derived Ngn2 neurons were cultured and transduced with the U6-T7 bearing lentivirus according to standard practice. Cell lines were fixed for 30 minutes with 4% PFA in IX PBS. Cell lines were immediately stained with their respective immunofluorescence labels at a 1/1000 dilution of both the primary and secondary antibodies. All following steps in the example were performed as written starting with permeabilization.
  • the method begins by contacting the cells with a construct that comprises a nucleic acid having the structure shown in FIG. 3A.
  • the cells have previously been transduced with a nucleic acid that encodes Cas9 at a high infection rate generating multiple integrants per cell.
  • Cells were selected with blasticidin (10 pg/mL) to remove non-infected cells.
  • the gRNA to be expressed in the cells of the sample are under control of the U6 promoter.
  • a second promoter, i.e. , the T7 promoter, for overexpression of the gRNA was introduced into the construct within the U6 promoter as shown in FIG. 3B.
  • the U6 promoter allows for the expression of the gRNAs in live cells, and the T7 promoter allows for the overexpression of the gRNA in fixed cells.
  • the sgRNA encoded by the construct that includes the two promoters can have the structure shown in Figure 7 of Dang et al. (2015) (e.g., the sgRNA can have an extended duplex (e.g., extended by 5 base pairs) that includes a mutation in the continuous sequence of Ts at position 4 (e.g., where the T at position 4 is mutated to C or G)).
  • Cells were transduced with lentivirus encoding the aforementioned vector at a low MOI to ensure on average 1 or fewer integrants per cell. Cells were than selected with puromycin (2ug/mL) to at least 95% purity prior to fixation.
  • the sample is then fixed in 4% Formaldehyde (5 ml 32% PF A, 4 ml lOx PBS and 31 ml H2O) for 30 minutes.
  • the fixed sample was then washed 3 times with PBS-T (PBS with 0.05% Tween) and permeabilized with 70% ethanol for 30 minutes.
  • PBS-T was then successively added and removed until the ethanol concentration was titrated below 1% to avoid sample dehydration.
  • the sample was then washed 3 times with PBS-Tween (PBS-T).
  • Decrosslinking was performed by incubating the sample in buffer including NaCl and sodium bicarbonate (8.4 ml H2O, 600 pL 5M NaCl and 1000 pL IM sodium bicarbonate) for 4 hours at 65°C and then washed 3 times with PBS-T.
  • FIG. 1 and FIG. 9A In vitro transcription of the gRNAs was performed using the T7 promoter located upstream of the gRNA nucleotide sequence as shown in FIG. 3A.
  • T7 in vitro transcription was performed by contacting the sample with a reaction mixture including a T7 RNA polymerase and NTP mix buffer from the HISCRIBE® T7 kit and the RNase inhibitor RiboLock as shown in Table 3. The in vitro transcription reaction was performed overnight at 37°C.
  • Reverse transcription was performed by contacting the sample with a reaction mixture including the reverse transcription primer oTK075, dNTPs, BSA, buffer, the Reverse Transcriptase (RT) of the RevertAid synthesis kit and the RNase inhibitor RiboLock as shown in Table 4.
  • the reverse transcription reaction was performed overnight at 37°C. Table 4
  • Gap filling was performed by contacting the sample with a reaction mixture including RNase H, BSA, the padlock probe oTK063, TaqIT polymerase, dNTPs, ampligase and an ampligase buffer as shown in Table 5. The reaction was run at 37°C for 5 minutes and then run at 45°C for 90 minutes. Table 5
  • RCA rolling circle amplification
  • Sequencing primer oTK064 (1 pM in 2X SSC buffer) was hybridized to the sample for 30 minutes at room temperature. The sample was then washed 3 times with PBS-T. A flat-top thermocycler was pre-warmed to 60°C. The incorporation mix (Illumina MiSeq reagent #1) was added to the sample at room temperature and the sample was moved to the flat-top thermocycler and incubated for 3 minutes at 60°C. The sample is then removed from the flat-top thermocycler. PR2 was added and then removed until the incorporation mix was diluted at least 50X. The sample was washed 3 times with PR2 at room temperature followed by 5 washes of PR2 at 60°C (5 minutes for each wash). Between each heated wash, the sample was removed from the thermocycler and the contents of the wells were replaced with fresh PR2.
  • DAPI solution 200 ng/mL DAPI in 2X SSC was added the sample (at least 2 mL/well for each well of a 6-well plate). DAPI solution was incubated with the sample for about 10 minutes to bind DNA. The samples were then imaged on a Nikon Ti-2 using NIS elements. A 545nm laser was used to excite Miseq G and T bases and a 638nm laser was used to excite Miseq A and C bases.
  • the conventional OPS method makes perturbations visible by converting expressed sgRNAs into sequences that can be read out by in situ sequencing through multiple enzymatic steps: reverse transcription, gap filling, and finally rolling circle amplification.
  • Each of these steps can be inefficient, which when combined with the low-expression of lentiviral delivered barcodes in many primary cell types (FIG. 10A) results in frequent experimental failures.
  • many primary cells express barcodes at relatively low levels (FIG. 10A)
  • Perturb View was developed, which enables facile optical detection of sgRNAs by utilizing T7 RNAP (T7 RNA polymerase) to amplify chromosomally integrated CRISPR perturbation vectors after cell fixation and phenotyping (FIG. 9A).
  • T7 RNAP The large molecular amplification of T7 RNAP produces a massive number of sgRNA molecules in the nucleus, which can be easily detected by in situ sequencing. This addresses the limitations of the conventional OPS protocol in detecting perturbations from cells with low levels of RNA barcode expression or barcode loss during phenotyping.
  • the barcodes can be robustly amplified and read through IVT and ISS in fixed cells.
  • Three vectors were tested: the CROP-seq vector, and two CROP-seq derivatives, where the T7 promoter was placed either upstream of or in place of the U6 promoter (FIG. 10B).
  • PF A paraformaldehyde
  • a decrosslinking step was added, followed by sgRNA overnight amplification with T7 RNAP.
  • chimeric promoters were designed, where the T7 sequence is embedded within the U6 promoter as close as possible to the sgRNA without disrupting CRISPR activity.
  • the published U6/T7 promoter design (Romanienko et al., PLoS One 11, e0148362 (2016); Binan et al., bioRxiv (2023) doi: 10.1101/2023.11.30.569494) resulted in substantial decreases in editing efficiency (FIG. 10C and FIG. 10H), necessitating the design of a new chimeric promoter to support highly efficient screens. 13 vector designs were tested, varying the location of the T7 promoter sequence as well as the leading/trailing nucleotides (Table 2).
  • the editing efficacy of the gRNAs was analyzed to determine if the incorporation of the T7 promoter into the U6 promoter (to generate chimeric promoters) affected the editing efficiency of the gRNAs in different cell lines.
  • the impact of the chimeric promoters on CRISPR editing efficiency of constructs targeting Target 1 on Target 1 expression levels was measured by flow in A549 cells, and several chimeric promoters were identified with identical CRISPR performance to the CROP-seq vector (FIG. 4 and FIG. 9B). As shown in FIG. 4, incorporation of the T7 promoter into the U6 promoter did not significantly affect the editing efficiency of a gRNA targeting Target 1 in A549 cells.
  • a gRNA under control of the T7-U6 promoter U6 r/ , U6 V2 , U6 V3 , U6 V4 , U6 1 , U6 , z ', U6 7 or U6 V8 exhibited the same efficiency as the gRNA under control of the U6 ,r/ promoter. It was noted that the truncated U6 variants based on the mini U6 promoter resulted in a decrease in editing activity (FIG. 9B).
  • chimeric promoters an optimal construct was selected that supported both efficient CRISPR activity and successful sgRNA detection by OPS after IVT (FIGS. 9A-B asterisk).
  • the chimeric promoters varied >40-fold in nuclear sgRNA production by IVT (FIG. 9C; 62 ⁇ 66 to 2,608 ⁇ l,898 AU; min to max mean nuclear signal ⁇ standard deviation (SD)), with AT -rich leading sequences and trailing G triplet crucial for optimal T7 activity (FIG. 9C).
  • the chosen construct (FIG. 9B, asterisk), used for all further Perturb View screens, had a 13 bp replacement from the original U6 promoter and high IVT efficiency (FIGS. 9C-D), while retaining CRISPR editing efficiency in all tested cell types (FIG. 9B, FIG. 10D)
  • gRNAs targeting Target 2 under the control the T7-U6 promoter U6 77 in IMPR90 fibroblasts and immortalized macrophages exhibited the same efficiency as the gRNAs under control of the U6 ,r/ promoter (FIG. 10D).
  • a targeting sgRNA was lentivirally transduced into Cas9 expressing cells, cells were kept in culture for 4 days and fixed in 4% paraformaldehyde in PBS for 30 min. Cells were washed twice with PBS and then incubated with fluorophore-conjugated antibody for staining a target membrane protein for Ih followed by additional PBS wash. The target expression level was measured by flow cytometry and plotted as a distribution with numbers indicating the fraction of knockout cells.
  • FIG. 5A shows that in situ sequencing using the disclosed method allows the product of T7 in vitro transcription to be visualized.
  • overexpression of the gRNA using the T7 promoter allows for the visualization of the gRNA in cells upon T7 addition (right panel) compared to standard ISS technique (left panel).
  • FIG. 5B shows that different T7-U6 promoters resulted in the overexpression of the gRNA for visualization of the gRNA in A549 cells. Similar results were shown in other cell types (FIG. 9H).
  • PerturbView across iPSC-derived neurons iPSC-derived neurons
  • iAstrocytes iPSC-derived astrocytes
  • IMR90 primary human fibroblasts
  • PerturbView showed comparable performance (FIG. 9H).
  • iNeurons macrophages and primary T cells, where conventional OPS performs poorly
  • PerturbView substantially improved barcode detection (1.6-, 2.9- and 2.0- fold, respectively, FIG. 9H).
  • FIG. 4 and FIGS. 9-10 these results are not target or cell specific as similar results were obtained using gRNAs targeting a different target gene in different cell lines. Accordingly, Perturb View enables screening in diverse cell types, in contrast to conventional OPS, which can be challenging to perform in noncancer cell lines due to poor sgRNA detection.
  • each cell was categorized in the experiment as follows: HA- positive cells with a detected targeting sgRNA are true positives; HA-positive cells with a non-targeting sgRNA or without a detected barcode are false negatives, HA-negative cells with targeting sgRNA are false positives, and HA-negative cells with non-targeting sgRNA or without a detected barcode are true negatives (FIG. 9E).
  • HA-positive cells with a detected targeting sgRNA are true positives
  • HA-positive cells with a non-targeting sgRNA or without a detected barcode are false negatives
  • HA-negative cells with targeting sgRNA are false positives
  • HA-negative cells with non-targeting sgRNA or without a detected barcode are true negatives.
  • Optical Pooled Screening is a versatile tool for dissecting biological processes.
  • its practical application has mostly been limited to low-plex phenotypes in cancer cell lines, due to challenges in efficiently and accurately detecting perturbation barcodes.
  • these limitations are addressed with Perturb View, a novel OPS approach that leverages IVT to amplify sgRNA barcodes, facilitating their detection across diverse cell types and tissue and their combination with different multiplexed phenotyping assays for RNA and proteins.
  • Perturb View signal is localized to the nucleus, making it easier to assign perturbations to challenging-to-segment cell types (e.g., neurons, fibroblasts) than conventional OPS.
  • the Perturb View vector was adapted from CROP-seq by replacing the U6 promoter with an engineered chimeric U6/T7 promoter, which enables IVT, while preserving CRISPR efficiency.
  • PerturbView cell libraries are also compatible with Perturb-Seq, enabling matched optical and molecular profiling.
  • the PerturbView screen of the NFKB pathway in BMDM cells is the first OPS in any primary cell. Because PerturbView has high detection efficiency, it significantly reduces the number of cells required for screening, a major consideration with primary cells. Moreover, compared to an earlier OPS of the NFKB pathway in a cancer cell line, the screen in primary immune cells shows key context-dependent biological differences, underscoring the importance of screening in a relevant biological context.
  • PerturbView facilitates multiplexed and multi-modal OPS, and paves the way for in vivo screens in animal models. While RNA and protein phenotyping before in situ sequencing compromised conventional OPS performance, in PerturbView, IVT was successfully executed post-phenotyping, thereby decoupling RNA detection from potential degradation and diffusion before OPS barcoding. In principle, any non-destructive in situ phenotyping method can be used prior to PerturbView, including highly-multiplexed techniques for protein detection, DNA and RNA FISH, and epigenetic modulators. Similarly, Perturb-View can be used for pooled tissue screens of CRISPR perturbations with spatial-omic readouts, which were previously hampered by inefficient RNA barcode detection (with ISS).
  • FISH-based detection of sgRNAs and barcodes has recently emerged as an alternative to ISS for reading out multiplex screens. These methods generally either directly detect an sgRNA with a FISH probe or detect an expanded FISH barcode often proximal to the sgRNA of interest. FISH methods excel at the rapid identification of barcode sequences but have several limitations: expanded FISH barcode proxies for sgRNA risk recombination during lentiviral production (resulting in high misidentification rates), and necessitate more complex cloning procedures; and directly probing against an sgRNA necessitates one FISH probe per sgRNA, severely limiting the multiplex capacity and potentially risking high crosstalk between FISH probes. In any case, it is anticipated that integrating the Perturb View construct design with FISH-based readouts will substantially improve the fidelity and speed of future FISH-based CRISPR screens.
  • Perturb View provides a key tool for optical pooled screening, with unprecedented sensitivity, accuracy and breadth, across cell lines, primary cells and issues in animal models.
  • Perturb View s compatibility with multiplexed phenotyping and spatial- omic readouts will enable comprehensive dissections of the impact of genetic perturbations and tracking of cell states across health and disease.
  • Example 2 Optical pooled screen in primary cells
  • NFKB signaling is a critical pathway that cells use to respond to stresses and stimuli. While OPS of the pathway has been performed in a cancer cell line, it is especially crucial in primary immune cells, and thus screening it in this native context may highlight distinct functions.
  • an NFKB translocation screen was performed in mouse bone marrow-derived macrophages (BMDMs) as shown in FIG. 11A using the detection method described in Example 1.
  • BMDMs mouse bone marrow-derived macrophages
  • This example shows that multi-modal screens can be performed on multiple immune signaling pathways in primary BMDMs using the presently disclosed method. As shown in FIGS. 7 and 11, this screen successfully recovered many of the known NFKB pathway components and discovered previously unknown components.
  • BMDMs were isolated from mice expressing Cas9-EGFP and cultured in DMEM high glucose, 10% FBS, glutamine, Pen/Strep, and 100 ng/ml CSF-1. After two days, these cells were split into new plates and infected with the lentivirus at low multiplicity of infection (MOI).
  • MOI multiplicity of infection
  • the library targets components of the canonical NFKB pathway, along with various other metabolic and signal transduction genes (Table 2).
  • the transduced BMDMs were stimulated with either TNFa, IL-ip, LPS or a FLO control for 40 minutes, fixed and stained for p65, and ISS was performed both by conventional OPS (without T7 IVT) and by Perturb View (with T7 IVT) (FIG. 11B, FIG. 121)
  • Conventional OPS efficiency was dramatically reduced by p65 phenotyping, even when using RNase inhibitors during staining and imaging (from 39% to 11%; FIG. 11B), while Perturb View after p65 immunofluorescence showed an 80% barcode detection rate (FIG. 11B).
  • Perturb View offers greater flexibility than conventional OPS, where ISS needs to be processed immediately after fixation. For example, phenotyping and genotyping using Perturb View a week post-fixation had comparable detection rates to those when genotyping is performed immediately after fixation (FIG. 11B, open vs. filled circles). sgRNA representation by Perturb View was highly concordant with that from next-generation sequencing of genomic DNA extracted from the cell library (FIG. 12A). Perturb View results were focused on for all further NFKB screens, scoring each gene (4 guides per gene) based on nuclear p65 levels (median of all guides per gene) (FIGS. 11C-11E).
  • This screen recovered established NFKB regulators as hits, including Ikbkb, Nfkbia, Tnfrsfla, Chuk, Nedd8, Tradd, Irak4, Myd88, Ripkl, Traf2, Traf6, Iraki, and Illrl, in the correct, stimulus-dependent manner, and captured both positive and negative regulators (which reduce or increase p65 signals when perturbed, respectively) (FIGS. 11C-11E). While some NFKB regulators played the same role in all three conditions, many others were context-dependent, playing a role only under relevant stimuli (FIGS. 11C-11E and FIG. 12B) For example, Tnfrsfla was the strongest hit for TNFa stimulation and Illrl for ILip stimulation; Myd88 and Irak4 mediated both ILip and LPS signaling but not TNFa signaling.
  • Map3k7 (Takl), a core NFKB component, only impacted p65 nuclear levels under LPS stimulation, but not under TNFa or ILip stimuli (FIG. 12C), suggesting that Map3k7 is only required for NFB activation in some contexts.
  • Prior screens conducted in human cancer cell lines showed that Map3k7 is necessary for both TNFa and ILip responses, whereas a screen in primary mouse bone-marrow-derived dendritic cells (BMDCs) showed that Map3k7 is dispensable for TNF protein production following LPS signaling in DCs. This highlights the context specificity even of key components of the NFKB pathway, in terms of both stimulus and cell type context, and thus the importance of Perturb View’s ability to screen in diverse primary cells.
  • Perturb View Although this screen is primarily designed for loss-of-function phenotypes (leading to a reduction in p65), Perturb View also identified several negative regulators (resulting in a nuclear p65 increase), a more challenging task®, given the already high p65 signals in the unperturbed, stimulated cells. These included Prkarla, where KO increased the response to ILip, and Tnfrsfla KO, which led to the expected loss of p65 signal under TNFa stimulus, but increased the p65 response in both LPS and ILip (FIGS. 12D-12E). Thus, Perturb View provided a generalizable, robust approach for image-based pooled screening across diverse, biologically relevant cell contexts.
  • Multiplexed imaging can provide systematic discovery tools for OPS screens, analogous to single-cell RNA-seq (scRNA-seq) profiles, but with the added richness of cell biological imaging phenotypes.
  • scRNA-seq single-cell RNA-seq
  • OPS has been limited to phenotyping with a few markers (up to 7 shown to date), requiring specialized staining and bleaching procedures and careful interleaving of the phenotypic assay with the ISS workflow.
  • PerturbView addresses this challenge by making it possible to perform all phenotyping steps upstream, prior to barcode RNA production from IVT and ISS.
  • PerturbView To demonstrate the compatibility of PerturbView with multiple high-plex immunofluorescence methods, multiple rounds of immunofluorescence staining of F4/80 in BMDM cells were conducted using either antibody stripping (4i) or fluorophore inactivation techniques (IBEX and cycIF) (FIG. 12J). OPS efficiency was severely compromised without PerturbView (FIG. 12J) None of the IF methods compromised PerturbView detection efficiency, supporting the idea that any non-destructive multiplexed phenotyping method is likely to be compatible with PerturbView.
  • FIG. 11F and FIG. 11 J This capability was leveraged to enhance the information content of the NFKB stimulation screens in BMDM with joint RNA and protein profiling. Specifically, after 24 hours of IL ip or TNFa stimulation of BMDMs (perturbed with the same library as before), the cells were fixed and HCR FISH was performed to measure three key transcripts (Gbp2, Ccl4 and Ripk2) followed by immunofluorescence for four proteins (Irfl, Nos2, p65 and phospho-rpS6).
  • genes are regulated by inflammatory stimuli, and were chosen to cover diverse functions, such as intracellular (Irfl, p65) signaling, intercellular (Ccl4) signaling and immunometabolism (Gbp2, Nos2 and phospho-rpS6).
  • the FISH probes were stripped with formamide treatment and bleached immunofluorescence signals with lithium borohydride prior to IVT and ISS (FIG. 11G). These approaches are compatible with high-plex profiling via further steps of iterative staining and signal quenching, paving the way to PerturbView screens with highly multiplexed molecular profiling readouts.
  • HI cluster 1
  • Birc2 and Birc3 impacted p65 translocation (FIGS. 11C-11D), suggesting that they mediate downstream immune signaling.
  • a module observed only in ILip signaling includes Atf3, Fos, Crebl, Junb and Pin: Atf/Creb family factors are known to interact with AP-1 proteins, such as Fos and Junb, and to regulate Pin.
  • a key metabolic module was also unique to ILip stimulation, including Ldha, Hspa4, Ddit3, Calr and Gck, which mediates mTOR and hypoxic signaling® and may reflect HIF la-mediated metabolic reprogramming during macrophage activation® after ILip stimulation.
  • each transcript and protein levels contribute to a cell’s phenotype was assessed.
  • the input features were restricted to only one of eight (each of the seven molecular species and DAPI), scored each of them for impact, and clustered perturbations by their score profiles across individual features. This allowed the identification of which phenotypic features yield similar perturbation scores, and which features contribute to the ‘impact’ call of each perturbation and in which condition (FIG. 12H ).
  • phospho-rpS6 was associated with the impact of many perturbations in Maf, Traf2, Ldha and Sod2, and quite distinct from all other phenotypic features.
  • Phospho-rpS6 was also associated with the impact of many perturbations in Tnfrsfla, Tradd, Nfkbia, Tnf, Srf and Birc3), and dropping the features related to phospho-rpS6 diminished the significance. In fact, we observed significant differences in median phospho-rpS6 level alone for 5 out of 6 of these gene perturbations (FDR ⁇ 0.05, FIG. 12K). In ILip stimulation, Ccl4 contributed to calling a separate set of impactful perturbations than Ripk2, Gbp2, or other features, consistent with a recent Perturb-seq study ⁇ reporting Ripk2 and Gbp2 as part of the same gene program, distinct from Ccl4. Taken together, Perturb View allows highly-multiplexed imaging screens with RNA and protein multiplex readouts in primary immune cells.
  • This screen demonstrates the utility of the present disclosed method to expand the scope of optical pooled screens to non-cancer cell lines.
  • an in situ optical screen in cancer cells transplanted into mice was performed using the method described in Example 1. This screen was performed to show that the presently disclosed method is compatible with read detection in a tissue section, enabling a diversity of in vivo screens with optical phenotypes.
  • an sgRNA library that included 100 barcoded (non-targeting) control sgRNAs was transduced into DLD-1 colorectal cancer cells using lentiviruses.
  • the transduced and selected DLD-1 colorectal cancer cells were then transplanted into mice to generate a xenograft tumor, and the tumor was collected to perform IVT and ISS in fresh- frozen and formalin-fixed paraffin-embedded (FFPE) sections.
  • FFPE paraffin-embedded
  • mice were anesthetized for manual restraint using Isoflurane gas in order to facilitate injection and minimize stress to the animal. Mice were placed in an Isoflurane gas induction chamber. As soon as they are immobile, they were removed from the chamber and placed on a nose cone delivering the anesthetic gas. The area of the injection site was cleaned with alcohol. Using forceps, the skin over the inoculation site was lifted and a sterile 25G needle attached to a 1.0 ml tuberculin syringe was inserted through the skin into the subcutaneous space. The inoculum dose of no more than 200 pl volume was deposited to inject the DLD- 1 cell suspensions. The needle was then held in place for approximately 3 seconds before being carefully withdrawn. The mouse was returned to its cage and monitored until fully recovered from anesthesia. The established tumors were sectioned on a slide as fresh-frozen or FFPE samples (FIG. 8).
  • PerturbView was combined with spatial transcriptomics, leveraging Xenium technology for fresh frozen tissue with a multi-tissue and cancer gene panel. Following the Xenium assay and DAPI staining, PerturbView was performed and sgRNA sequences were captured over six cycles of ISS (FIG. 13E). PerturbView successfully produced joint RNA- sgRNA maps. After the Xenium run, -100% of ISS reads were mapped with high fidelity (within ⁇ 2 hamming distance) to sgRNA barcodes (FIG. 13B). Leiden clustering of the transcriptome identified four clusters in all samples, which were strongly associated with spatial patterns (FIG. 13J). Two of the clusters from each sample have low transcriptome counts, which are likely to be necrotic or mouse cells (FIG. 13J).
  • the spatial PerturbView was leveraged to decipher intra- and inter-clone expression heterogeneity of the cancer cells in vivo, hypothesizing that cells of the same clonal origin will be more similar, due to both cell memory and microenvironment similarity.
  • non-targeting sgRNAs function as high diversity clonal barcodes (descendants of transplanted cells all expressed the same sgRNA)
  • a clonal map was generated, in which cells carrying the same barcodes and locally clustered together were considered as a clone (FIG. 13F).
  • High consistency in the clonal map was also observed between adjacent sections (FIG. 14D).
  • tissue regions with high barcode mapping rate were categorized into low (bottom 25 th percentile), middle (25 th -75 th percentile) and high clonal diversity (upper 75 th percentile) (by Shannon’s diversity index, FIG. 13G and FIG. 13K), and performed differential expression analysis between the expression profiles of cells with matched sgRNAs in low versus high diversity regions.
  • Hallmark epithelial -to-mesenchymal (EMT) transition markers 21 such as PDGFRB, BASP1, COL5A2, TNC, BASP1 were higher in clones in low entropy regions, while apical junction genes 21 (COL17A1, VWF, GDF15), usually considered epithelial, were higher in clones in high entropy regions and intestinal stem cell marker LGR5 was found to be particularly upregulated in regions of high clonal diversity.
  • EMT Hallmark epithelial -to-mesenchymal

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Abstract

La présente divulgation concerne des procédés améliorés d'imagerie d'acides nucléiques cibles par séquençage in situ. En particulier, la présente divulgation concerne des constructions d'acides nucléiques (et des compositions de celles-ci), des procédés, des systèmes et des kits pour imager au moins un acide nucléique cible dans un seul échantillon à l'aide d'un séquençage in situ.
PCT/US2024/061106 2023-12-20 2024-12-19 Imagerie optique d'acides nucléiques Pending WO2025137335A1 (fr)

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