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WO2024243040A2 - Lecture multiomique à haut débit d'arn et d'adn génomique dans des cellules uniques - Google Patents

Lecture multiomique à haut débit d'arn et d'adn génomique dans des cellules uniques Download PDF

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Publication number
WO2024243040A2
WO2024243040A2 PCT/US2024/029950 US2024029950W WO2024243040A2 WO 2024243040 A2 WO2024243040 A2 WO 2024243040A2 US 2024029950 W US2024029950 W US 2024029950W WO 2024243040 A2 WO2024243040 A2 WO 2024243040A2
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sequence
seq
cell
gdna
cells
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WO2024243040A3 (fr
WO2024243040A8 (fr
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Lars M. STEINMETZ
Dominik LINDENHOFER
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Europaisches Laboratorium fuer Molekularbiologie EMBL
Leland Stanford Junior University
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Europaisches Laboratorium fuer Molekularbiologie EMBL
Leland Stanford Junior University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • DNA, RNA and protein can be currently read-out individually or in a combinatorial fashion.
  • Combined protein readout is mostly achieved by using oligo- linked antibodies that recognize cell membrane proteins, which in turn limits the type and number of proteins that can be assessed in addition to either RNA or DNA (15, 16).
  • RNA and genomic DNA are laborious and low-throughput (1, 2, 18).
  • Current approaches are microtiter plate well-based and RNA and DNA is either split before downstream analysis or processed and tagged without separation. In all cases the number of cells retrieved after these methods is very low, which confounds interpretation of the data significantly, makes is unusable for highthroughput screening purposes or other large-scale experimental approaches.
  • More scalable methods like the recently published method of Olsen et al., 2023, uses nucleosome depletion and Tn5-based tagmentation to enable simultaneous gDNA and RNA read-out in single cells (3).
  • Tn5-based approaches have the limitation that not all the gDNA within a single cell gets tagmented at each locus, limiting the coverage per given target site.
  • these methods need to read-out the entire tagmented gDNA, which means very high sequencing costs.
  • This present disclosure describes a method that enables read out of a multitude of RNA transcripts and gDNA loci simultaneously within single cells in a targeted fashion with high coverage in all cells. It has numerous potential applications that involve linking genomic information to transcriptomic signatures.
  • the disclosure provides methods and compositions that are useful for simultaneously detecting RNA and genomic DNA (gDNA) within the same cell.
  • the disclosure provides a method for simultaneously detecting RNA and genomic DNA (gDNA) within the same cell, comprising: (i) providing a single cell suspension comprising fixed and permeabilized cells; (ii) performing an in-situ reverse transcription (RT) step to generate cDNA molecules by contacting the cells with reverse transcriptase and a RT primer; (iii) lysing the cells in a first droplet comprising a first reverse primer that hybridizes to the cDNA molecules and a second reverse primer that hybridizes to the gDNA molecules, wherein the first or second reverse primer comprises a R2N or R2 overhang sequence; (iv) fusing the first droplet to a second droplet comprising PCR reagents and a forward primer that hybridizes to both the cDNA and gDNA molecules and comprises a capture sequence (CS
  • the single cell suspension is fixed with paraformaldehyde (PFA) or glyoxal.
  • PFA paraformaldehyde
  • the R2N overhang sequence comprises the nucleic acid sequence GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG (SEQ ID NO:2), and the R2 overhang sequence comprises the nucleic acid sequence GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO:3).
  • the RT primer comprises a capture sequence (CS), a sample barcode (SBC) sequence, a unique molecular identifier (UMI) sequence, and a sequence that binds to mRNA.
  • the CS sequence hybridizes to a complementary sequence attached to a solid support
  • the SBC sequence comprises a known sequence of variable length
  • the UMI sequence comprises a random sequence of variable length.
  • the CS comprises the nucleic acid sequence GTACTCGCAGTAGTC (SEQ ID NO:1).
  • the SBC sequence is from 4 to 50 nucleotides in length.
  • the SBC sequence comprises a nucleic acid sequence selected from the group consisting of TCGCCTTA (SEQ ID NO:4), CTAGTACG (SEQ ID NO:5), TTCTGCCT (SEQ ID NO:6), GCTCAGGA (SEQ ID NO:7), AGGAGTCC (SEQ ID NO:8), CATGCCTA (SEQ ID NO:9), GTAGAGAG (SEQ ID NO:10), and CCTCTCTG (SEQ ID NO:11).
  • the UMI sequence is from 4 to 50 nucleotides in length.
  • the UMI sequence comprises the nucleic acid sequence NNNNNN (SEQ ID NO:12).
  • the sequence that binds to mRNA comprises oligo(dT) or a sequence that hybridizes to any region of the mRNA.
  • step (v) of the method comprises PCR conditions that favor binding of forward and reverse primers to the cDNA and gDNA molecules, thereby producing a first set of PCR products each comprising the CS.
  • the second droplet further comprises cell barcode oligonucleotides each comprising a cell barcode (CBC) and a sequence complementary to the CS, thereby the first set of PCR products hybridize to the cell barcode oligonucleotides, producing a second set of PCR products each comprising the CS and the CBC.
  • the cell barcode oligonucleotides are attached to a solid support in the second droplet prior step (iv). In some embodiments, the cell barcode oligonucleotides are released from the solid support after the second droplet is fused with the first droplet.
  • simultaneous amplification in step (v) produces a cDNA library and a gDNA library.
  • the method further comprises sequencing the cDNA and gDNA libraries.
  • the cDNA library is sequenced by a first sequencing modality
  • the gDNA library is sequenced by a second sequencing modality.
  • the first and second sequencing modalities are the same or different.
  • the first sequencing modality comprises scRNA-seq and the second sequencing modality comprises scDNA-seq.
  • the first sequencing modality comprises Illumina® next generation sequencing (NGS), and the second sequencing modality comprises Nextera® NGS, or the first sequencing modality comprises Nextera® NGS, and the second sequencing modality comprises Illumina® NGS.
  • step (iii) of the method comprises contacting the cells with proteinase K to lyse the cells.
  • the cell is a prokaryotic or eukaryotic cell.
  • the cell is an induced pluripotent stem cell (iPSC).
  • iPSC induced pluripotent stem cell
  • the cell is a genetically modified cell.
  • the cell is modified using a CRISPR/Cas gene editing system.
  • the CRISPR/Cas gene editing system is CRISPR interference (CRISPRi).
  • CRISPRi CRISPR interference
  • RNA expressed by the genetically modified cell is sequenced by scRNA-seq and genomic DNA is sequenced by scDNA-seq.
  • the disclosure provides a reaction mixture comprising RNA and gDNA from a single cell, reverse transcriptase, and a RT primer comprising a capture sequence (CS), a sample barcode (SBC) sequence, a unique molecular identifier (UMI) sequence, and a sequence that binds to mRNA.
  • CS capture sequence
  • SBC sample barcode
  • UMI unique molecular identifier
  • the CS hybridizes to a complementary sequence attached to a solid support
  • the SBC sequence comprises a known sequence of variable length
  • the UMI sequence comprises a random sequence of variable length
  • the sequence that binds to mRNA comprises oligo(dT) or a sequence that hybridizes to any region of the mRNA.
  • the CS comprises the nucleic acid sequence GTACTCGCAGTAGTC (SEQ ID NO:1).
  • the SBC sequence is from 4 to 50 nucleotides in length.
  • the SBC sequence comprises a nucleic acid sequence selected from the group consisting of TCGCCTTA (SEQ ID NO:4), CTAGTACG (SEQ ID NO:5), TTCTGCCT (SEQ ID NO:6), GCTCAGGA (SEQ ID NO:7), AGGAGTCC (SEQ ID NO:8), CATGCCTA (SEQ ID NO:9), GTAGAGAG (SEQ ID NO:10), and CCTCTCTG (SEQ ID NO:11).
  • the UMI sequence is from 4 to 50 nucleotides in length.
  • the UMI sequence comprises the nucleic acid sequence NNNNNN (SEQ ID NO:12).
  • the reaction mixture comprises cDNA molecules produced by reverse transcription of the RNA and a reverse primer that hybridizes to both the cDNA and the gDNA molecules and comprises a R2N or R2 overhang sequence.
  • the reverse primer comprising the R2N overhang sequence comprises the nucleic acid sequence GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG (SEQ ID NO:2) and the reverse primer comprising the R2 overhang sequence comprises the nucleic acid sequence GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO:3).
  • the reaction mixture comprises a, one or more, or a plurality of polynucleotides selected from a sequence in any one of SEQ ID Nos: 1-83.
  • the disclosure provides a polynucleotide comprising a capture sequence (CS).
  • the polynucleotide further comprises a sample barcode (SBC) sequence, a unique molecular identifier (UMI) sequence, and/or a sequence that binds to mRNA.
  • SBC sample barcode
  • UMI unique molecular identifier
  • the SBC sequence of the polynucleotide comprises a known sequence of variable length, and the UMI sequence of the polynucleotide comprises a random sequence of variable length.
  • the polynucleotide comprises a sequence in any one of SEQ ID Nos: 1-83.
  • the CS can comprise the nucleic acid sequence GTACTCGCAGTAGTC (SEQ ID NO:1).
  • the SBC sequence is from 4 to 50 nucleotides in length.
  • the SBC sequence can comprise a nucleic acid sequence selected from the group consisting of TCGCCTTA (SEQ ID NO:4), CTAGTACG (SEQ ID NO:5), TTCTGCCT (SEQ ID NO:6), GCTCAGGA (SEQ ID NO:7), AGGAGTCC (SEQ ID NO:8), CATGCCTA (SEQ ID NO:9), GTAGAGAG (SEQ ID NO:10), and CCTCTCTG (SEQ ID NO:11).
  • the UMI sequence is from 4 to 50 nucleotides in length.
  • the UMI comprises the nucleic acid sequence NNNNNNNN (SEQ ID NO:12).
  • the sequence that binds to mRNA can comprise oligo(dT) or a sequence that hybridizes to any region of the mRNA.
  • Fig. 1 Overview of targeted scDNA-scRNA-seq method with high sensitivity read- out. Main steps involve fixation of a single-cell suspension and in-situ reverse transcription (RT) followed by a multiplexed PCR within individual droplets. Both cDNA and gDNA targets are amplified at the same time. UMI, unique molecular identifier; SBC, sample barcode; CS, capture sequence; CBC, cell barcode. [0038] Figs.
  • 2A-2I scDNA-scRNA-seq proof-of principle experiment.
  • A Outline of experiment. Fixation conditions tested are PFA and Glyoxal. 28 gDNA and 30 cDNA targets were amplified.
  • B Ranked cell barcodes. Automatic threshold is indicated.
  • C Number of cells found per fixation condition. Number of gDNA (D) and cDNA (G) targets found per cell. Number of gDNA (E) and cDNA (H) target reads or UMIs/cell. gDNA (F) or cDNA (I) target coverage per cell. [0039] Figs. 3A-3H. Expression/Coverage of cDNA and gDNA targets and comparison to bulk RNA-seq data.
  • gDNA Average expression of gDNA (A) and cDNA (B) targets. Size indicates fraction of cells target is expressed/found in; Color indicates expression (log10).
  • C Comparison of expressed cDNA targets to bulk RNA-seq data. Z-score, data is scaled by column.
  • D Clustered based on expressed cDNA targets. Example UMAPs of gDNA (E, F) and cDNA (G, H) targets. [0040] Figs. 4A-4E. CRSIPRi perturbation screen using scDNA-scRNA-seq.
  • A Modification of the technology to enable separate sequencing of both cDNA and gDNA libraries.
  • R2 overhangs for cDNA and gDNA are distinct (Illumina and Nextera, respectively) enabling separate NGS library generation and sequencing.
  • B Screen target selection. Number of non-targeting control (NTC), CRISPRi control and eQTL gRNAs is shown. cDNA and gDNA targets amplified are indicated.
  • C Experimental outline. Lentiviral CROP-seq gRNA library is used to infect transgenic iPSCs that express CRISPRi from the AAVS1 locus.
  • D gRNA coverage. Color indicated type of gRNA.
  • E Screening results. NTC, CRISPRi control and eQTL gRNAs are shown.
  • Figs. 5A-5G Editing screen using scDNA-scRNA-seq.
  • A Screen target selection. Number of coding controls (introducing STOP codons) and eQTL pegRNAs is shown. cDNA and gDNA targets amplified are indicated.
  • B Experimental outline. pegRNA library is used to transfect two transgenic iPSCs that express PEmax or PEmax and MLH1dn from the AAVS1 locus.
  • C Editing efficiencies for both PEmax cell lines shown.
  • D Dimplot indicating clusters found.
  • the oligonucleotide is attached to the barcoding bead by a photocleavable linker (PhotoC-E), and comprises an R1N sequence, followed by a first cell barcode (CBC1, – 9 bp), an off-staggered constant space linker (CSL,– 14 to 17 bp), a second cell barcode (CBC2, – 9 bp) and a capture sequence (CS).
  • CBC1, – 9 bp first cell barcode
  • CSL off-staggered constant space linker
  • CBC2, – 9 bp second cell barcode
  • CS capture sequence
  • RNA and gDNA targets are amplified at the same time.
  • UMI unique molecular identifier
  • BC barcode.
  • B Outline of proof-of-concept (POP) experiment. Fixation conditions and number or gDNA/RNA targets are indicated.
  • C Knee plot of ranked lineages by sequencing depth.
  • D Number of cells found per fixation condition.
  • E Correct sample BC detection per cell. Reads for maximum sample BC found was divided by the total amount of reads for all RNA targets found per cell.
  • Figs. 8A-8H Quality controls and comparison of PFA and glyoxal fixation conditions.
  • a-d Quality control plots before (A, B) and after (C, D) filtering for low quality cells. Color indicates either fraction of detected gDNA or RNA targets.
  • e, f gDNA (E) or RNA (F) coverage and detection.
  • g, h Comparison of coverage and detection between PFA and Glyoxal for each gDNA (G) and RNA (H) target.
  • Figs. 9A-9D UMAPs and clustering of POP data.
  • A Clustering of POP SDR-seq data.
  • Clusters are indicated.
  • B Color coding of clusters in UMAP by fixation condition.
  • C UMAP plots of ubiquitously expressed genes GAPDH, POU5F1 and SOX2.
  • D UMAP plots of cluster-specifically expressed genes KLF5, SALL4 and ESRRB.
  • Figs. 10A-10J SDR-seq is amendable for hundreds of targets simultaneously.
  • A Outline of panel size testing experiments. gDNA and RNA targets are equal within panels; Shared targets are indicated.
  • B, C Pearson correlation of detection (B) and coverage (C) of shared gDNA targets between panels.
  • D, E Pearson correlation of detection (B) and coverage (C) of shared genes between panels.
  • F Outline of chromatin sites tested.
  • OEG overlapping expressed gene. NOEG, non-overlapping expressed gene.
  • PLS promotor-like sequence.
  • pELS proximal enhancer-like sequence.
  • dELS distal enhancer-like sequence.
  • G Detection of different chromatin sites between panels. Size indicates fraction of cells detected in. Color indicates read coverage.
  • H Outline of expression levels tested.
  • I Detection of genes with different expression levels between panels. Size indicates fraction of cells detected in. Color indicates read coverage.
  • J Heatmap of expression of all shared genes between panels. Z- score, data is scaled by row. [0047] Figs. 11A-11F. SDR-seq with separate library generation for RNA and gDNA targets.
  • FIG. 12A-12J Metrics for target detection and coverage across differently sized target panels.
  • B gDNA coverage and detection for all targets across panels tested.
  • C gDNA target detection per cell for all targets across panels tested.
  • D gDNA coverage and detection for shared targets across panels tested.
  • E gDNA target detection per cell for shared targets across panels tested.
  • F Quality metrics of panel size experiment. Color indicates fraction of RNA targets/cell recovered.
  • G RNA coverage and detection for all targets across panels tested.
  • H RNA target detection per cell for all targets across panels tested.
  • I RNA coverage and detection for shared targets across panels tested.
  • J RNA target detection per cell for shared targets across panels tested. [0049] Figs.13A-13B.
  • FIG.14A-14K Comparison of target detection and coverage across differently sized target panels.
  • A, B Comparison of coverage and detection for shared targets across panels tested for each gDNA (A) and RNA (B) target.
  • Figs.14A-14K SDR-seq is sensitive to detect gene expression changes and link them to variants.
  • A Outline of the CRISPRi screen. NTC, non-targeting control.
  • B Experimental outline of the CRISPRi screen.
  • C Volcano plot for CRISPRi screen with different gRNA classes indicating foldchange and P-value. Significant hits (P-value ⁇ 0.05) are colored. For NTCs all genes measured are shown. For other gRNA classes only the intended target for each gRNA is shown.
  • FIG. 15A-15E Quality metrics for CRISPRi screen and functional testing of PE iPSCs.
  • A Overview of gRNA assignment for CRISPRi screen.
  • B Coverage for each gRNA in CRISPRi screen.
  • C Relative distance of gRNA binding site to transcription start site (TSS). Positive values are after TSS (within transcript), negative values before transcript. Size indicates P-value.
  • FIG. 16A-16E Editing efficiency and genotyping in PE screen.
  • A Editing efficiency in PE screen.
  • CRISPRi is indicated as a control.
  • B Editing efficiency for each locus that was assessed. Loci that were either HET or ALT in the PE iPCSs are not shown. Color indicates either PE cell line or CRISPRi.
  • FIG.17A-17B POU5F1 locus in BE screen.
  • A Intended edit is shown at POU5F1 locus and its impact on gene expression.
  • B All measured variants shown for POU5F1 with their impact on gene expression.
  • about means a range extending to +/- 10% of the specified value (e.g., +/- 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the specified value). In embodiments, about means the specified value.
  • a “barcode” refers to a nucleotide sequence that is used to identify an individual group of polynucleotides and distinguish it from other groups of polynucleotides among a mixture of groups.
  • sample barcode refers to a unique barcode for a sample (e.g., a biological sample, a tissue, a biopsy, a cell) that is different from the barcode sequence associated with other samples (e.g., a biological sample, a tissue, a biopsy, a cell).
  • cell barcode or “CBC” refers to a unique barcode for a cell that is different from the barcode sequence associated with other cells.
  • UMI unique molecular identifier
  • UMIs may be sequenced along with the DNA sequences with which they are associated to identify sequencing reads that are from the same source nucleic acid.
  • the term “UMI” is used herein to refer to both the nucleotide sequence of the UMI and the physical nucleotides, as will be apparent from context.
  • UMIs may be random, pseudo-random, or partially random, or nonrandom nucleotide sequences that are inserted into adapters or otherwise incorporated in source nucleic acid (e.g., DNA or RNA) molecules to be sequenced.
  • source nucleic acid e.g., DNA or RNA
  • each UMI is expected to uniquely identify any given source molecule present in a sample.
  • a probe can further include a cleavage domain and/or a functional domain (e.g., a primer-binding site, such as for next generation sequencing (NGS).
  • NGS next generation sequencing
  • the capture sequence has a length of 10 to 100 nucleotides, 10 to 80 nucleotides, 10 to 60 nucleotides, 10 to 40 nucleotides, 20 to 40 nucleotides, or 15 to 35 nucleotides.
  • the capture sequence comprises random nucleic acid sequences for capturing total RNA or genomic DNA sequence.
  • the capture sequence comprises specific target nucleic acid sequences for capturing target RNA or DNA sequences.
  • the terms “overhang sequence” refers to a nucleic acid sequence that is located at the 5’ end of an oligonucleotide or primer that, at least initially, does not hybridize to the target sequence that is amplified during the first round of primer extension.
  • the term “genome editing” refers to a type of genetic engineering in which DNA is inserted, replaced, or removed from a target DNA (e.g., the genome of a cell) using one or more nucleases and/or nickases.
  • the nucleases create specific double-strand breaks (DSBs) at desired locations in the genome and harness the cell's endogenous mechanisms to repair the induced break by homology-directed repair (HDR) (e.g., homologous recombination) or by nonhomologous end joining (NHEJ).
  • HDR homology-directed repair
  • NHEJ nonhomologous end joining
  • two nickases can be used to create two single-strand breaks on opposite strands of a target DNA, thereby generating a blunt or a sticky end.
  • Any suitable DNA nuclease can be introduced into a cell to induce genome editing of a target DNA sequence.
  • reverse transcriptase refers to its plain and ordinary meaning as an enzyme used to generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription.
  • polypeptide and “protein” refer to a polymer of amino acid residues and are not limited to a minimum length. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition.
  • polypeptide refers to a protein which includes modifications, such as deletions, additions and substitutions to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, as through site directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
  • sequence specific endonuclease refers to an enzyme that cleaves at a specific sequence within a polynucleotide sequence.
  • the nuclease activity can be partially or completed inhibited, so that only one of the two strands or neither strand is cleaved,.
  • sequence specific endonucleases include CRISPR associated (Cas) nuclease, a Zinc-finger nuclease, a Transcription activator-like effector nuclease (TALEN), or a meganuclease.
  • Cas9 encompasses type II clustered regularly interspaced short palindromic repeats (CRISPR) system of Cas9 endonucleases from any species, and also includes biologically active fragments, variants, analogs, and derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double- strand breaks).
  • CRISPR clustered regularly interspaced short palindromic repeats
  • a Cas9 endonuclease binds to and cleaves DNA at a site comprising a sequence complementary to its bound guide RNA (gRNA).
  • gRNA bound guide RNA
  • a Cas9 polynucleotide, nucleic acid, oligonucleotide, protein, polypeptide, or peptide refers to a molecule derived from any source. The molecule need not be physically derived from an organism but may be synthetically or recombinantly produced. Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database.
  • NCBI National Center for Biotechnology Information
  • sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein, wherein the variant retains biological activity, such as Cas9 site-directed endonuclease activity. See also Fonfara et al. (2014) Nucleic Acids Res.
  • the term refers only to the primary structure of the molecule.
  • the term includes triple-, double- and single-stranded DNA, as well as triple- , double- and single-stranded RNA. It also includes modifications, such as by methylation and/or by capping, and unmodified forms of the polynucleotide.
  • polynucleotide examples include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D- ribose), any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, Oreg., as Neugene) polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA.
  • PNAs peptide nucleic acids
  • polynucleotide oligonucleotide
  • nucleic acid nucleic acid molecule
  • these terms include, for example, 3 ⁇ -deoxy-2',5 ⁇ -DNA, oligodeoxyribonucleotide N3 ⁇ P5 ⁇ phosphoramidates, 2'-O-alkyl-substituted RNA, double- and single-stranded DNA, as well as double- and single-stranded RNA, microRNA, DNA:RNA hybrids, and hybrids between PNAs and DNA or RNA, and also include known types of modifications, for example, labels which are known in the art, methylation, "caps," substitution of one or more of the naturally occurring nucleotides with an analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine
  • an analog e.g., 2-aminoadenosine, 2-thiothymidine
  • the term also includes locked nucleic acids (e.g., comprising a ribonucleotide that has a methylene bridge between the 2'-oxygen atom and the 4'-carbon atom).
  • locked nucleic acids e.g., comprising a ribonucleotide that has a methylene bridge between the 2'-oxygen atom and the 4'-carbon atom.
  • identity refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100.
  • nucleic acid and/or amino acid sequences include sequences that are at least 90% identical to a nucleic acid sequence disclosed herein, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%, identical to a nucleic acid and/or amino acid sequence disclosed herein as determined by the Smith and Waterman algorithm.
  • complementary refers to polynucleotides that are able to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in an anti-parallel orientation between polynucleotide strands. Complementary polynucleotide strands can base pair in a Watson- Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil (U) rather than thymine (T) is the base that is considered to be complementary to adenosine.
  • uracil when a uracil is denoted in the context of the present disclosure, the ability to substitute a thymine is implied, unless otherwise stated.
  • “Complementarity” may exist between two RNA strands, two DNA strands, or between a RNA strand and a DNA strand. It is generally understood that two or more polynucleotides may be “complementary” and able to form a duplex despite having less than perfect or less than 100% complementarity.
  • Two sequences are "perfectly complementary” or “100% complementary” if at least a contiguous portion of each polynucleotide sequence, comprising a region of complementarity, perfectly base pairs with the other polynucleotide without any mismatches or interruptions within such region.
  • Two or more sequences are considered “perfectly complementary” or “100% complementary” even if either or both polynucleotides contain additional non-complementary sequences as long as the contiguous region of complementarity within each polynucleotide is able to perfectly hybridize with the other.
  • "Less than perfect" complementarity refers to situations where less than all of the contiguous nucleotides within such region of complementarity are able to base pair with each other.
  • a gRNA may comprise a sequence "complementary" to a target sequence (e.g., major or minor allele), capable of sufficient base-pairing to form a duplex (i.e., the gRNA hybridizes with the target sequence). Additionally, the gRNA may comprise a sequence complementary to a sequence adjacent to a PAM sequence, wherein the gRNA also hybridizes with the sequence adjacent to a PAM sequence in a target DNA.
  • a "target site” or “target sequence” is the nucleic acid sequence recognized (i.e., sufficiently complementary for hybridization) by a guide RNA (gRNA) or a homology arm of a donor polynucleotide.
  • the target site may be allele-specific (e.g., a major or minor allele).
  • target edit site or “target edit locus” or “edit locus” refer to a target site in the host cell genome comprising a nucleic acid sequence recognized by a guide RNA (gRNA) or a homology arm of a donor polynucleotide that is or was edited by the methods of the disclosure.
  • Recombinant as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature.
  • the term "recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide.
  • the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expresses the foreign gene to produce the protein under expression conditions.
  • transformation refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion. For example, direct uptake, transduction or f-mating are included.
  • the exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.
  • Recombinant host cells refers to cells which can be, or have been, used as recipients for recombinant vector or other transferred DNA, and include the original progeny of the original cell which has been transfected.
  • a "coding sequence” or a sequence which "encodes" a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or "control elements").
  • the boundaries of the coding sequence can be determined by a start codon at the 5 ⁇ (amino) terminus and a translation stop codon at the 3 ⁇ (carboxy) terminus.
  • a coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences.
  • a transcription termination sequence may be located 3 ⁇ to the coding sequence.
  • the coding sequence may be interrupted by introns which can be self- splicing group I or group II introns or those which are spliced out by the host cell splicing machinery,
  • Typical "control elements” include, but are not limited to, transcription promoters, transcription enhancer elements, introns (located anywhere in the transcript), transcription termination signals, polyadenylation sequences (located 3 ⁇ to the translation stop codon), sequences for optimization of initiation of translation (located 5 ⁇ to the coding sequence), and translation termination sequences.
  • "Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present.
  • the promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.
  • "Expression cassette” or "expression construct” refers to an assembly which is capable of directing the expression of the sequence(s) or gene(s) of interest.
  • An expression cassette generally includes control elements, as described above, such as a promoter which is operably linked to (so as to direct transcription of) the sequence(s) or gene(s) of interest, and often includes a polyadenylation sequence as well.
  • the expression cassette described herein may be contained within a plasmid or viral vector construct (e.g., a vector for genome modification comprising a genome editing cassette comprising a promoter operably linked to a polynucleotide encoding a guide RNA and a donor polynucleotide).
  • the construct may also include, one or more selectable markers, a signal which allows the construct to exist as single stranded DNA (e.g., a M13 origin of replication), at least one multiple cloning site, and a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication) or “yeast” origin of replication (e.g. a 2-micron vector or centromeric vector with an autonomously replicating sequence (ARS)).
  • a signal which allows the construct to exist as single stranded DNA
  • a M13 origin of replication e.g., a M13 origin of replication
  • a “mammalian" origin of replication e.g., a SV40 or adenovirus origin of replication
  • yeast origin of replication e.g. a 2-micron vector or centromeric vector with an autonomously replicating sequence (ARS)
  • ARS autonomously replicating sequence
  • transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981) Gene 13:197.
  • Such techniques can be used to introduce one or more exogenous nucleic acids moieties into suitable host cells.
  • the term refers to both stable and transient uptake of the genetic material and includes uptake of peptide- or antibody-linked nucleic acids.
  • a “vector” is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes).
  • target cells e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes.
  • vector construct e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes.
  • expression vector e transfer vector
  • the term includes cloning and expression vehicles, as well as plasmid and viral vectors.
  • variant refer to modifications of a nucleic acid and/or amino acid sequence disclosed herein.
  • a nucleic acid sequence of the disclosure can include a variant having one, two, three, four, five or more nucleotide substitutions of the reference sequence.
  • a variant of a nucleic acid sequence of the disclosure can hybridize to a sequence that is complementary to the original reference sequence.
  • variant also refer biologically active derivatives of the reference molecule that retain desired activity, such as site-directed Cas9 endonuclease activity.
  • variants and analogs refer to compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature) and/or deletions, relative to the native molecule, so long as the modifications do not destroy biological activity and which are "substantially identical" to the reference molecule as defined below.
  • amino acid sequences of such variants and analogs will have a high degree of sequence identity to the reference sequence, e.g., an amino acid sequence having greater than or equal to 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, when the two sequences are aligned.
  • variants and analogs will include the same number of amino acids but will include substitutions, as explained herein.
  • the term "mutein” further includes polypeptides having one or more amino acid-like molecules including but not limited to compounds comprising only amino and/or imino molecules, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring (e.g., synthetic), cyclized, branched molecules and the like.
  • the term also includes molecules comprising one or more N-substituted glycine residues (a "peptoid") and other synthetic amino acids or peptides.
  • amino acids are generally divided into four families: (1) acidic -- aspartate and glutamate; (2) basic -- lysine, arginine, histidine; (3) non-polar -- alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar -- glycine, asparagine, glutamine, cysteine, serine threonine, and tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.
  • the polypeptide of interest may include up to about 5-10 conservative or non-conservative amino acid substitutions, or even up to about 15-25 conservative or non-conservative amino acid substitutions, or any integer between 5-25, so long as the desired function of the molecule remains intact.
  • Gene transfer or “gene delivery” refers to methods or systems for reliably inserting DNA or RNA of interest into a host cell. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of host cells.
  • Gene delivery expression vectors include, but are not limited to, vectors derived from bacterial plasmid vectors, viral vectors, non-viral vectors, adenoviruses, retroviruses, alphaviruses, pox viruses, and vaccinia viruses.
  • heterologous refers to biological material that is introduced, inserted, or incorporated into a recipient (e.g., host) organism that originates from another organism. Typically, the heterologous material that is introduced into the recipient organism (e.g., a host cell) is not normally found in that organism.
  • Heterologous material can include, but is not limited to, nucleic acids, amino acids, peptides, proteins, and structural elements such as genes, promoters, and cassettes.
  • a host cell can be, but is not limited to, a bacterium, a yeast cell, a mammalian cell, or a plant cell.
  • the introduction of heterologous material into a host cell or organism can result, in some instances, in the expression of additional heterologous material in or by the host cell or organism.
  • the transformation of a yeast host cell with an expression vector that contains DNA sequences encoding a bacterial protein may result in the expression of the bacterial protein by the yeast cell.
  • the incorporation of heterologous material may be permanent or transient.
  • heterologous material may be permanent or transient.
  • Methods for simultaneously detecting RNA and genomic DNA (gDNA) within the same cell [0089] Described herein are compositions and methods for simultaneously detecting RNA and genomic DNA (gDNA) within the same cell.
  • the compositions and methods can be used for multiplexed polymerase chain reaction (PCR) and cell barcoding of PCR fragments within droplets.
  • PCR polymerase chain reaction
  • the instant disclosure provides the following advantages. First, the inventors have demonstrated that targeted read-out of both transcriptomic and gDNA is feasible using the methods of the disclosure. Second, separate library construction enables sequencing using different sequencing modalities and coverage. Third, the methods of the disclosure are sensitive enough to allow detection of gene expression changes and is therefore amendable for screening purposes.
  • the scale of the methods described herein (10,000 cells in one experiment) is a significant improvement over existing microtiter plate well-based methods (2), while coverage of targets is better than in splitseq based approaches (3).
  • the instant methods have the advantage of directly reading out the loci of interest in a targeted fashion having high sensitivity and cost-effective sequencing.
  • the method for simultaneously detecting RNA and genomic DNA (gDNA) within the same cell comprises providing a single cell suspension as a starting material for obtaining genomic DNA and RNA molecules.
  • the single cell suspension can comprise fixed and permeabilized cells, which allows enzymes and oligonucleotides to enter the cells which maintain cell membrane integrity.
  • RNA molecules in the cell can then be contacted with a reverse transcriptase, dNTPs and a primer (e.g., an RT primer) in a container to generate cDNA molecules by in-situ reverse transcription (RT) of the RNA molecules in the cell.
  • a primer e.g., an RT primer
  • the RT primer comprises a sequence that binds to mRNA.
  • the sequence that binds to mRNA comprises oligo(dT) or a sequence that hybridizes to any region of the mRNA.
  • the RT primer further comprises a sequence selected from the group consisting of a unique molecular identifier (UMI) sequence, an extension sequence for primer binding, a sample barcode (SBC) sequence, a capture sequence (CS), and combinations thereof.
  • the CS provides an overhang sequence that is incorporated into a double stranded PCR product (see below) and is complementary to a sequence attached to a solid support, such as a barcoded bead.
  • the RT primer comprises a capture sequence (CS), an extension sequence for primer binding, a sample barcode sequence, a unique molecular identifier (UMI) sequence, and a poly dT sequence.
  • the RT primer comprises, from 5’ to 3’, a CS with an optional extension sequence for an optimal primer binding site, a SBC sequence, a UMI sequence, and a sequence that binds to mRNA (e.g., oligo(dT) or a sequence that hybridizes to any region of the mRNA).
  • the RT primer comprises a nucleic acid sequence selected from Table 1 (SEQ ID NOs:14-21).
  • the CS comprises the nucleic acid sequence GTACTCGCAGTAGTC (SEQ ID NO:1).
  • the CS hybridizes to a complementary sequence attached to a solid support described herein.
  • the complementary sequence attached to the solid support further comprises a cell barcoding sequence.
  • the SBC sequence comprises a known sequence of variable length.
  • the SBC sequence is from 4 to 50 nucleotides in length, e.g., 4 to 50, 4 to 40, 4 to 30, 4 to 20, 4 to 15, 4 to 14, 4 to 13, 4 to 12 nucleotides in length, any sub- range between 4 and 50 nucleotides in length, or 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides in length.
  • the SBC comprises a sequence selected from the group consisting of TCGCCTTA (SEQ ID NO:4), CTAGTACG (SEQ ID NO:5), TTCTGCCT (SEQ ID NO:6), GCTCAGGA (SEQ ID NO:7), AGGAGTCC (SEQ ID NO:8), CATGCCTA (SEQ ID NO:9), GTAGAGAG (SEQ ID NO:10), and CCTCTCTG (SEQ ID NO:11).
  • the UMI sequence comprises a random sequence of variable length.
  • the UMI sequence is from 4 to 50 nucleotides in length, e.g., 4 to 50, 4 to 40, 4 to 30, 4 to 20, 4 to 15, 4 to 14, 4 to 13, 4 to 12 nucleotides in length, any sub- range between 4 and 50 nucleotides in length, or 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides in length.
  • the UMI sequence comprises the nucleic acid sequence NNNNNN (SEQ ID NO:12), where N is any nucleotide.
  • the extension sequence for primer binding comprises the sequence GACACGTC (SEQ ID NO:29).
  • the container for the RT reaction can be any suitable space for holding a liquid volume that is isolated from other containers, including but not limited to a well of a microtiter plate or a microfluidic droplet.
  • the fixed and permeabilized cells are lysed in a reaction mixture comprising a second primer (e.g., a reverse primer) that hybridizes to both the cDNA and gDNA molecules and contains either a R2N or R25’ overhang sequence.
  • the reaction mixture can be present in a separate (second) container, such as a microfluidic droplet (e.g., a first droplet).
  • the reaction mixture comprising the cell lysate and reverse primer can then be combined with PCR reagents and two different forward primers, where one forward primer hybridizes to the cDNA molecules and the second forward primer hybridizes to the gDNA molecules.
  • the forward primer comprises a capture sequence (CS) 5’ overhang sequence.
  • the forward primer that hybridizes to the cDNA molecules comprises a 5’ CS overhang and a sample barcode (SBC) sequence.
  • the SBC sequences comprises a known sequence of variable length.
  • the SBC sequence is from 4 to 50 base-pairs in length, e.g., 4 to 50, 4 to 40, 4 to 30, 4 to 20, 4 to 15, 4 to 14, 4 to 13, or 4 to 12 base-pairs in length, or any sub-range between 4 and 50 base-pairs in length.
  • the SBC comprises a sequence selected from the group consisting of TCGCCTTA (SEQ ID NO:4), CTAGTACG (SEQ ID NO:5), TTCTGCCT (SEQ ID NO:6), and GCTCAGGA (SEQ ID NO:7).
  • the forward primer that hybridizes to the gDNA molecules comprises a 5’ CS overhang and a gDNA specific sequence.
  • Reverse transcription of mRNA in the cell produces first strand cDNA molecules that can then be amplified with appropriate forward and reverse primers.
  • the gDNA present in the same cell can also be amplified with appropriate forward and reverse primers.
  • the amplification reaction can take place in a separate or different container or droplet than the RT reaction.
  • the forward and/or reverse primers used to amplify the cDNA and gDNA molecules comprise the same 5’ sequence region and a different 3’ sequence region that hybridizes to complementary sequences in the cDNA or gDNA.
  • the forward primer used to amplify cDNA comprises a 5’ CS and a 3’ SBC sequence
  • the reverse primer used to amplify cDNA comprises a 5’ R2N sequence and a 3’ sequence that hybridizes to complementary sequences in the cDNA.
  • the forward primer used to amplify cDNA comprises a sequence selected from the group consisting of SEQ ID NOs: 22-25.
  • the reverse primer used to amplify cDNA comprises GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG (SEQ ID NO:2).
  • the forward primer used to amplify cDNA comprises a 5’ CS and a 3’ extension sequence for primer binding
  • the reverse primer used to amplify cDNA comprises a 5’ R2 sequence and a 3’ sequence that hybridizes to complementary sequences in the cDNA.
  • the forward primer used to amplify cDNA comprises GTACTCGCAGTAGTCGACACGTC (SEQ ID NO:30)
  • the reverse primer used to amplify cDNA comprises GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO:3) and a 3’ sequence that hybridizes to complementary sequences in the cDNA.
  • the forward primer used to amplify gDNA comprises a 5’ CS and a 3’ sequence that hybridizes to complementary sequences in the gDNA
  • the reverse primer used to amplify gDNA comprises a 5’ R2N and a 3’ sequence that hybridizes to complementary sequences in the gDNA.
  • the forward primer used to amplify gDNA comprises SEQ ID NO:1 and a 3’ sequence that hybridizes to complementary sequences in the gDNA.
  • the reverse primer used to amplify gDNA comprises SEQ ID NO:2 and a 3’ sequence that hybridizes to complementary sequences in the gDNA.
  • the second droplet further comprises one or more cell barcode oligonucleotides.
  • Each cell barcode oligonucleotide comprises a cell barcode (CBC) sequence and a sequence complementary to the CS. Therefore, when the first set of PCR products are generated within the second droplet, the PCR products bind to the cell barcode oligonucleotides in the hybridization of the CS, thereby introducing the CBC to the PCR products, resulting in a second set of PCR products.
  • each second set of PCR product comprises the CS and the CBC.
  • the cell barcode oligonucleotides are attached to a solid support.
  • the PCR reagents and forward primer are present in a second microfluidic droplet that is fused or merged with the first droplet comprising the reaction mixture comprising the cell lysate and reverse primer. Exemplary embodiments are illustrated in Figs.1 and 7a. [0105] Exemplary sequences useful in the practice of the methods are provided in Table 1 below. Table 1. Exemplary sequences for amplifying cDNA and gDNA from the same cell.
  • the container comprising the PCR reagents comprises a solid support attached to oligonucleotides comprising sequences that are complementary to and hybridize to the reverse-complement of the CS sequence present in the double-stranded PCR product.
  • the oligos attached to the solid support comprise an R1N sequence (that is useful for Illumina® based sequencing), a cell barcode sequence, and a UMI sequence.
  • the oligos attached to the solid support comprise an R1N sequence (that is useful for Illumina® based sequencing), a first cell barcode sequence (CBC1), an off-staggered constant space linker (CSL), a second cell barcode sequence (CBC2), and a capture sequence (CS).
  • the off-staggered CSL can be a variable-length nucleic acid sequence.
  • the CSL has a length of 5 to 50 bp, 10 to 40 bp, 10 to 30 bp, or 10 to 20 bp, 20 to 40 bp, or 15 to 35 bp. In some embodiments, the CSL has a length of 14 to 17 bp.
  • the off-staggered CSL separates the two CBCs for better sequencing quality.
  • the CS facilitates capture target RNAs or gDNAs for cell barcoding.
  • Exemplary sequences attached to the solid support are provided in Table 2 below. Table 2. Exemplary sequences attached to solid support.
  • the solid support is a bead, such as a gel bead.
  • the oligonucleotides are removed from the bead prior to the PCR reaction.
  • Exemplary beads include those provided by 10X Genomics (San Francisco, CA) and Mission Bio (South San Francisco, CA). An exemplary barcoded bead is shown in Fig.6.
  • the first step comprises contacting fixed and permeabilized cells with reverse transcriptase, dNTPs, and a reverse transcription primer to generate cDNA (“reverse transcription step”).
  • reverse transcriptase reverse transcriptase
  • dNTPs reverse transcriptase
  • reverse transcription primer reverse transcription primer
  • Two microfluidic droplets are then generated consecutively and targeted multiplexed PCR is performed in each droplet for both the cDNA and gDNA targets.
  • the first droplet contains Proteinase K and one or more reverse primers.
  • in the first droplet cells from the RT step are lysed and different reverse primers comprising a R2N 5’ overhangs are used to amplify the cDNA targets or the gDNA targets.
  • the reverse primers used to amplify the cDNA targets comprise a R2N 5’ overhang sequence and a cDNA specific sequence.
  • the reverse primers used to amplify the gDNA targets comprise a R2N 5’ overhang sequence and a gDNA specific sequence.
  • the second drop is produced by fusing or combining the first droplet with a droplet comprising PCR reagents, a forward primer comprising a CS 5’ overhang for amplifying the target cDNA or gDNA sequence, and a solid support attached to oligonucleotides comprising sequences that are complementary to and hybridize to the reverse-complement of the CS sequence present in the double-stranded PCR product.
  • PCR reagents typically include a thermostable polymerase and dNTPs.
  • the oligonucleotides attached to the solid support comprises a cell barcode sequence.
  • the solid support is referred to as a cell barcoding bead.
  • the method is modified to enable separate sequencing of both cDNA and gDNA libraries from the same single cell.
  • the first droplet comprises a first plurality of reverse primers comprising a R25’ overhang and a cDNA specific sequence that is used to amplify each cDNA target, and a second plurality of reverse primers comprising a R2N 5’ overhang and a gDNA specific sequence that is used to amplify each gDNA target.
  • the first droplet also contains reagents for lysing cells, such as proteinase-K, following the RT step.
  • the first droplet is fused or combined with a second droplet comprising PCR reagents, a forward primer comprising a CS overhang for amplifying the cDNA and gDNA sequences, and a solid support attached to oligonucleotides comprising sequences that are complementary to and hybridize to the reverse-complement of the CS sequence present in the double-stranded PCR product.
  • the oligonucleotides attached to the solid support comprise a cell barcode sequence.
  • the solid support comprises a bead, and the oligonucleotides attached to the bead comprise a photocleavable linker (see exemplary oligonucleotides in Fig.
  • the oligonucleotides attached to the bead can be contacted with UV light, thereby releasing the oligonucleotides from the bead surface into solution.
  • releasing the oligonucleotides from the bead into solution may increase the PCR efficiency.
  • suitable beads are provided by Mission Bio, Inc. (South San Francisco, CA).
  • each droplet contains only 1 bead on average in order to provide single cell resolution.
  • the solid support comprises a bead that is capable of being dissolved by heat, and the oligonucleotides attached to the bead can be released from the bead surface into solution by heating the droplet during the second droplet step.
  • suitable beads are provided by 10X Genomics (Pleasanton, CA).
  • Screening of Genetically Modified Cells [0113]
  • the methods of the disclosure further comprise screening cDNA and gDNA from genetically modified (e.g., genetically edited) cells.
  • the cells are genetically modified using CRISPR/Cas technology.
  • the cells are genetically modified using CRISP interference (“CRISPRi”).
  • the CRISPRi system uses a catalytically inactive Cas9 (dCas9) protein (e.g., comprising two point mutations D10A and H840A) that lacks endonuclease activity to regulate genes in an RNA-guided manner, and can be used to block transcription of target genes without cutting the target DNA.
  • dCas9- single guide RNA (sgRNA) complex binds to DNA elements complementary to the sgRNA and causes a steric block that halts transcript elongation by RNA polymerase, resulting in repression of the target gene.
  • the CRISPRi system comprises a dCas9 fused to repressor proteins (e.g., SALL1 and SDS3), and a guide RNA specifically designed to target the region immediately downstream of a gene’s transcriptional start site (TSS).
  • the guide RNA associates with dCas9-SALL1-SDS3 and directs the repressor complex to the DNA target site.
  • the guide RNAs are designed to target the transcription start site of genes.
  • the guide RNAs are designed to target sequences that are transcribed into mRNA (referred to as “cDNA targets”).
  • the cells are fixed and permeabilized as described above, and the cells are contacted with reverse transcriptase, dNTPS, and a RT primer to produce cDNA.
  • the RT primer comprises one or more of a CS sequence, an extension sequence for primer binding, a sample barcode sequence, and a poly dT sequence.
  • the cells are added to a first droplet comprising a first reverse primer comprising a CS sequence and R2 overhang that is used to amplify each cDNA target and a second reverse primer comprising a CS sequence and R2N overhang that is used to amplify each gDNA target.
  • the first droplet also contains reagents for lysing cells, such as proteinase-K.
  • the first droplet is the fused or combined with a second droplet comprising PCR reagents, a forward primer comprising a CS overhang for amplifying the cDNA and gDNA target sequences, and a solid support attached to oligonucleotides and a solid support attached to oligonucleotides comprising sequences that are complementary to and hybridize to the reverse-complement of the CS sequence present in the double-stranded PCR product.
  • the oligonucleotides attached to the solid support comprises a cell barcode sequence.
  • the amplified cDNA and gDNA libraries can then be sequenced to determine RNA expression levels of the targeted genes.
  • the cDNA and gDNA libraries are sequenced using next generation sequencing (Illumina®).
  • the RNA-guided nuclease e.g., dCas9
  • a nucleic acid encoding the RNA- guided nuclease such as an RNA (e.g., messenger RNA) or DNA (expression vector). Codon usage may be optimized to improve production of an RNA-guided nuclease in a particular cell or organism.
  • a nucleic acid encoding an RNA-guided nuclease can be modified to substitute codons having a higher frequency of usage in a yeast cell, a bacterial cell, a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence.
  • the protein can be transiently, conditionally, or constitutively expressed in the cell.
  • Donor polynucleotides and gRNAs are readily synthesized by standard techniques, e.g., solid phase synthesis via phosphoramidite chemistry, as disclosed in U.S. Patent Nos. 4,458,066 and 4,415,732, incorporated herein by reference; Beaucage et al., Tetrahedron (1992) 48:2223-2311; and Applied Biosystems User Bulletin No. 13 (1 April 1987).
  • Other chemical synthesis methods include, for example, the phosphotriester method described by Narang et al., Meth. Enzymol. (1979) 68:90 and the phosphodiester method disclosed by Brown et al., Meth. Enzymol. (1979) 68:109.
  • gRNA-donor polynucleotide cassettes can be produced by standard oligonucleotide synthesis techniques and subsequently ligated into vectors. Moreover, libraries of gRNA-donor polynucleotide cassettes directed against thousands of genomic targets can be readily created using highly parallel array-based oligonucleotide library synthesis methods (see, e.g., Cleary et al. (2004) Nature Methods 1:241-248, Svensen et al. (2011) PLoS One 6(9):e24906).
  • adapter sequences can be added to oligonucleotides to facilitate high- throughput amplification or sequencing.
  • a pair of adapter sequences can be added at the 5 ⁇ and 3 ⁇ ends of an oligonucleotide to allow amplification or sequencing of multiple oligonucleotides simultaneously by the same set of primers.
  • restriction sites can be incorporated into oligonucleotides to facilitate cloning of oligonucleotides into vectors.
  • oligonucleotides comprising gRNA-donor polynucleotide cassettes can be designed with a common 5 ⁇ restriction site and a common 3 ⁇ restriction site to facilitate ligation into the genome modification vectors.
  • a restriction digest that selectively cleaves each oligonucleotide at the common 5 ⁇ restriction site and the common 3 ⁇ restriction site is performed to produce restriction fragments that can be cloned into vectors (e.g., plasmids or viral vectors), followed by transformation of cells with the vectors comprising the gRNA-donor polynucleotide cassettes.
  • vectors e.g., plasmids or viral vectors
  • a restriction site can also be added in between the gRNA and donor polynucleotide sequences to enable a second cloning step for the introduction of a guide RNA scaffold sequence or other constructs into the vector.
  • Amplification of polynucleotides encoding gRNA-donor polynucleotide cassettes may be performed, for example, before ligation into genome modification vectors or before sequencing and after barcoding. Any method for amplifying oligonucleotides may be used, including, but not limited to polymerase chain reaction (PCR), isothermal amplification, nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), strand displacement amplification (SDA), and ligase chain reaction (LCR).
  • the genome editing cassettes comprise common 5 ⁇ and 3 ⁇ priming sites to allow amplification of the gRNA-donor polynucleotide sequences in parallel with a set of universal primers.
  • Genome editing may be performed on a single cell or a population of cells of interest and can be performed on any type of cell, including any cell from a prokaryotic, eukaryotic, or archaeon organism, including bacteria, archaea, fungi, protists, plants, and animals.
  • Cells from tissues, organs, and biopsies, as well as recombinant cells, genetically modified cells, cells from cell lines cultured in vitro, and artificial cells (e.g., nanoparticles, liposomes, polymersomes, or microcapsules encapsulating nucleic acids) may all be used in the practice of the present disclosure.
  • the methods of the disclosure are also applicable to editing of nucleic acids in cellular fragments, cell components, or organelles comprising nucleic acids (e.g., mitochondria in animal and plant cells, plastids (e.g., chloroplasts) in plant cells and algae).
  • Cells may be cultured or expanded prior to or after performing genome editing as described herein.
  • the cells are induced pluripotent stem cells (iPSCs).
  • iPSCs induced pluripotent stem cells
  • the Cas9 nuclease and gRNAs can be delivered to cells in vitro or in vivo using any method known in the art.
  • a DNA expression vector comprising a nucleic acid sequence encoding the Cas9 protein operably linked to transcriptional and/or translation control elements such as promoters and terminators is transfected into cells.
  • an mRNA molecule encoding the Cas9 protein is transfected into cells.
  • purified Cas9 protein is delivered to the cell cytoplasm or nucleus, for example by encapsulating the protein in a vesicle.
  • Suitable methods include using viral and nonviral vectors and physical methods.
  • Viral vectors include lentiviral, adenovirus (AV), adeno-associated virus (AAV) and retroviral vectors.
  • Physical methods include microinjection and electroporation of DNA, RNA or protein, cell penetrating peptides, lipofection, lipid-based nanoparticles comprising DNA, RNA or protein, and gold based nanoparticles.
  • Other suitable methods include virus-like particles, extracellular vesicles, transposon systems, chemically- induced transformation, typically using divalent cations (e.g., CaCl 2 ), microinjection. See, e.g., Yip BH. Recent Advances in CRISPR/Cas9 Delivery Strategies.
  • the gRNAs or libraries thereof are delivered into cells using a viral vector, such as a lentiviral vector. In some embodiments, the gRNAs or libraries thereof are delivered into cells using lipofection.
  • the gRNAs are prime editing guide RNAs (pegRNAs) that are extended on the 3’ end to install edits using a prime editing system.
  • the prime editing system comprises a modified version of the Cas9 enzyme fused with reverse transcriptase.
  • Compositions Reaction Mixtures [0123]
  • the disclosure provides reaction mixtures that are useful for amplifying cDNA and gDNA from the same cell.
  • Reaction mixtures typically include a nucleic acid template (e.g., RNA or DNA), an RNA or DNA polymerase, one or more oligo nucleotide primers, and dNTPs.
  • the reaction mixture comprises RNA and gDNA from a single cell, reverse transcriptase, and a RT primer comprising a sequence that binds to mRNA.
  • the sequence that binds to mRNA comprises oligo(dT) or a sequence that hybridizes to any region of the mRNA.
  • the RT primer further comprises a sequence selected from the group consisting of a unique molecular identifier (UMI) sequence, a sample barcode (SBC) sequence, a capture sequence (CS), and combinations thereof.
  • UMI unique molecular identifier
  • SBC sample barcode
  • CS capture sequence
  • the RT primer comprises, from 5’ to 3’, a CS, a SBC sequence, a UMI sequence, and a sequence that binds to mRNA (e.g., oligo(dT) or a sequence that hybridizes to any region of the mRNA).
  • the RT primer comprises, from 5’ to 3’, a CS, an extension sequence for primer binding, a SBC sequence, a UMI sequence, and a sequence that binds to mRNA (e.g., oligo(dT) or a sequence that hybridizes to any region of the mRNA).
  • the CS comprises the nucleic acid sequence GTACTCGCAGTAGTC (SEQ ID NO:1).
  • the SBC sequence comprises a known sequence of variable length.
  • the SBC sequence is from 4 to 50 nucleotides in length, e.g., 4 to 50, 4 to 40, 4 to 30, 4 to 20, 4 to 15, 4 to 14, 4 to 13, 4 to 12 nucleotides in length, any sub- range between 4 and 50 nucleotides in length, or 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides in length.
  • the SBC sequences comprises a known sequence of variable length.
  • the SBC comprises a sequence selected from the group consisting of TCGCCTTA (SEQ ID NO:4), CTAGTACG (SEQ ID NO:5), TTCTGCCT (SEQ ID NO:6), GCTCAGGA (SEQ ID NO:7), AGGAGTCC (SEQ ID NO:8), CATGCCTA (SEQ ID NO:9), GTAGAGAG (SEQ ID NO:10), and CCTCTCTG (SEQ ID NO:11).
  • the UMI sequence comprises a random sequence of variable length.
  • the UMI sequence is from 4 to 50 nucleotides in length, e.g., 4 to 50, 4 to 40, 4 to 30, 4 to 20, 4 to 15, 4 to 14, 4 to 13, 4 to 12 nucleotides in length, any sub- range between 4 and 50 nucleotides in length, or 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides in length.
  • the UMI sequence comprises the nucleic acid sequence NNNNNN (SEQ ID NO:12), where N is any nucleotide.
  • the extension sequence for primer binding comprises the sequence GACACGTC (SEQ ID NO:29).
  • the forward and/or reverse primers used to amplify the cDNA and gDNA molecules comprise the same 5’ sequence region and a different 3’ sequence region that hybridizes to complementary sequences in the cDNA or gDNA.
  • the forward primer used to amplify cDNA comprises a 5’ CS and a 3’ SBC sequence
  • the reverse primer used to amplify cDNA comprises a 5’ R2N sequence and a 3’ sequence that hybridizes to complementary sequences in the cDNA.
  • the forward primer used to amplify cDNA comprises a sequence selected from the group consisting of SEQ ID NOs:22-25.
  • the reverse primer used to amplify cDNA comprises SEQ ID NO:2 and a 3’ sequence that hybridizes to complementary sequences in the cDNA.
  • the forward primer used to amplify cDNA comprises a 5’ CS and a 3’ extension sequence for primer binding
  • the reverse primer used to amplify cDNA comprises a 5’ R2 sequence and a 3’ sequence that hybridizes to complementary sequences in the cDNA.
  • the forward primer used to amplify cDNA comprises GTACTCGCAGTAGTCGACACGTC (SEQ ID NO:30), and the reverse primer used to amplify cDNA comprises SEQ ID NO:3 and a 3’ sequence that hybridizes to complementary sequences in the cDNA.
  • the forward primer used to amplify gDNA comprises a 5’ CS and a 3’ sequence that hybridizes to complementary sequences in the gDNA
  • the reverse primer used to amplify gDNA comprises a 5’ R2N and a 3’ sequence that hybridizes to complementary sequences in the gDNA.
  • the forward primer used to amplify gDNA comprises SEQ ID NO:1 and a 3’ sequence that hybridizes to complementary sequences in the gDNA.
  • the reverse primer used to amplify gDNA comprises SEQ ID NO:2 and a 3’ sequence that hybridizes to complementary sequences in the gDNA.
  • the reaction mixture comprises cDNA molecules produced by reverse transcription of the RNA and a reverse primer that hybridizes to both the cDNA and the gDNA molecules and comprises a R2N or R2 overhang sequence.
  • the reverse primer comprising the R2N overhang sequence comprises the nucleic acid sequence GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG (SEQ ID NO:2) and the reverse primer comprising the R2 overhang sequence comprises the nucleic acid sequence GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT SEQ ID NO:3).
  • the reaction mixture comprises a polynucleotide of the disclosure. In some embodiments, the reaction mixture comprises a (one or more) polynucleotide(s) of any one of SEQ ID Nos: 1-83. Polynucleotides [0134] In another aspect, the disclosure provides polynucleotides that are useful in the methods of the disclosure.
  • the polynucleotide can be used to prime reverse transcription of an mRNA molecule.
  • the polynucleotide comprises a capture sequence (CS).
  • the CS comprises the nucleic acid sequence GTACTCGCAGTAGTC (SEQ ID NO:1).
  • the polynucleotide further comprises a sample barcode (SBC) sequence, a unique molecular identifier (UMI) sequence, and/or a sequence that binds to mRNA.
  • the SBC sequence comprises a known sequence of variable length
  • the UMI sequence comprises a random sequence of variable length.
  • the SBC sequence is from 4 to 50 nucleotides in length.
  • the UMI sequence is from 4 to 50 nucleotides in length, e.g., 4 to 50, 4 to 40, 4 to 30, 4 to 20, 4 to 15, 4 to 14, 4 to 13, 4 to 12 nucleotides in length, any sub-range between 4 and 50 nucleotides in length, or 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides in length.
  • the SBC sequence comprises a nucleic acid sequence selected from the group consisting of TCGCCTTA (SEQ ID NO:4), CTAGTACG (SEQ ID NO:5), TTCTGCCT (SEQ ID NO:6), GCTCAGGA (SEQ ID NO:7), AGGAGTCC (SEQ ID NO:8), CATGCCTA (SEQ ID NO:9), GTAGAGAG (SEQ ID NO:10), and CCTCTCTG (SEQ ID NO:11).
  • the UMI sequence is from 4 to 50 nucleotides in length.
  • the UMI sequence is from 4 to 50 nucleotides in length, e.g., 4 to 50, 4 to 40, 4 to 30, 4 to 20, 4 to 15, 4 to 14, 4 to 13, 4 to 12 nucleotides in length, any sub-range between 4 and 50 nucleotides in length, or 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides in length.
  • the UMI comprises the nucleic acid sequence NNNNNN (SEQ ID NO:12).
  • the sequence that binds to mRNA comprises oligo(dT) or a sequence that hybridizes to any region of the mRNA.
  • the polynucleotide comprises a sequence in any one of SEQ ID Nos: 1-83.
  • the polynucleotide comprises a capture sequence (CS), a sample barcode (SBC) sequence, a unique molecular identifier (UMI) sequence, and nucleic acid sequence that binds to mRNA.
  • the polynucleotide comprises SEQ ID NO:1.
  • the polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NOs: 5-11, SEQ ID NO:12, and SEQ ID NO:13.
  • the polynucleotide comprises, from 5’ to 3’, a CS and a sample barcode (SBC) sequence.
  • the polynucleotide comprises SEQ ID NOs:1 and 4.
  • the polynucleotide comprises SEQ ID NOs:1 and 5.
  • the polynucleotide comprises SEQ ID NOs:1 and 6.
  • the polynucleotide comprises SEQ ID NOs:1 and 7.
  • the polynucleotide comprises SEQ ID NOs:1 and 8.
  • the polynucleotide comprises SEQ ID NOs:1 and 9.
  • the polynucleotide comprises SEQ ID NOs:1 and 10. In some embodiments, the polynucleotide comprises SEQ ID NOs:1 and 11. In some embodiments, the polynucleotide comprises a nucleic acid sequence selected from a sequence in Table 3. Table 3. [0139] In some embodiments, the polynucleotide comprises, from 5’ to 3’, a CS, an SBC sequence, and a UMI. In some embodiments, the polynucleotide comprises SEQ ID NOs:1, 4 and 12. In some embodiments, the polynucleotide comprises SEQ ID NOs:1, 5 and 12. In some embodiments, the polynucleotide comprises SEQ ID NOs:1, 6 and 12.
  • the polynucleotide comprises SEQ ID NOs:1, 7 and 12. In some embodiments, the polynucleotide comprises SEQ ID NOs:1, 8 and 12. In some embodiments, the polynucleotide comprises SEQ ID NOs:1, 9 and 12. In some embodiments, the polynucleotide comprises SEQ ID NOs:1, 10 and 12. In some embodiments, the polynucleotide comprises SEQ ID NOs:1, 11 and 12. In some embodiments, the polynucleotide comprises a nucleic acid sequence selected from a sequence in Table 4. Table 4. [0140] In some embodiments, the polynucleotide comprises an extension sequence for primer binding.
  • polynucleotide comprises the sequence GACACGTC (SEQ ID NO:29). In some embodiments, the polynucleotide comprises, from 5’ to 3’, a CS and an extension sequence for primer binding. In some embodiments, the polynucleotide comprises SEQ ID NOs:1 and 29 (GTACTCGCAGTAGTCGACACGTC (SEQ ID NO:30)). [0141] In some embodiments, the polynucleotide comprises, from 5’ to 3’, a CS, an extension sequence for primer binding, and an SBC sequence. In some embodiments, the polynucleotide comprises a nucleic acid sequence selected from a sequence in Table 5. Table 5.
  • the polynucleotide comprises, from 5’ to 3’, a CS, an extension sequence for primer binding, an SBC sequence, and a UMI sequence. In some embodiments, the polynucleotide comprises a nucleic acid sequence selected from a sequence in Table 6. Table 6. [0143] In some embodiments, the polynucleotide comprises, from 5’ to 3’, a CS, an extension sequence for primer binding, a SBC sequence, a UMI sequence, and a sequence that binds to an mRNA. In some embodiments, the polynucleotide comprises a nucleic acid sequence selected from a sequence in Table 7. Table 7. EXAMPLES Example 1.
  • Cells were spun at 500 g for 3 min at 4 C, supernatant removed, resuspended in 300 ⁇ l of 0.5x PBS with 0.2 U/ ⁇ l RNasin® Plus Ribonuclease Inhibitor, filtered through a 40 ⁇ m strainer, counted and diluted to 1.4 x10 6 cells/ml.
  • Reverse transcription master mix consisting of a final concentration of 1x RT Buffer, 0.25 U/ ⁇ l Enzymatics RNAse Inhibitor (Biozym – 180520), 0.2 U/ ⁇ l RNasin® Plus Ribonuclease Inhibitor, 500 mM dNTPs and 20 U/ ⁇ l Maxima H Minus Reverse Transcriptase (ThermoFisher - EP0752) was prepared on ice for in 8 ⁇ l for a total reaction volume of 20 ⁇ l. 4 ⁇ l of reverse transcription oligo (12.5 ⁇ M) were combined in each 96-well plate with 8 ⁇ l reverse transcription master mix.
  • In-situ RT processed cell pellet from previous step was resuspended in the cell buffer of Mission Bio, cells were counted and diluted to the appropriate concentration of 3000- 4000 cells/ ⁇ l.
  • Custom primers were used in the multiplexed droplet PCR amplification step.
  • cDNA primers were designed using the TAPseq primer prediction tool with at targeted Tm of 60 °C (Schraivogel et al., 2020).
  • gDNA primers were designed using the Tapestri Designer (see the internet at designer.missionbio.com). Version 1 gDNA and cDNA both had CS and R2N overhangs. Version 2 gDNA primers had CS and R2N, whereas cDNA primers had CS and R2 overhangs.
  • RNA and gDNA simultaneously within the same cell in a targeted fashion. This is achieved by combining in-situ reverse transcription (RT) (4) with a multiplexed PCR within droplets (Fig. 1).
  • RT reverse transcription
  • Fig. 1 a first proof-of-principle we used the commercial droplet generation device of the Tapestri platform from Mission Bio. Cells are dissociated into a single-cell suspension and fixed followed by several washes and a permeabilization step to enable enzymes to enter the cells while maintaining cell membrane integrity. This is followed by an in-situ RT step generating cDNA.
  • Primers used for this RT step contain oligo(dT)s to bind to polyadenylated mRNAs, a unique molecular identifier (UMI), a sample barcode (SBC) that can be used for different experimental conditions and a capture sequence (CS) that is used to introduce a cell barcode (CBC) in subsequent droplet steps.
  • UMI unique molecular identifier
  • SBC sample barcode
  • CS capture sequence
  • CBC cell barcode
  • first droplet generation cells are treated with proteinase K yielding a cell lysate and reverse primer is added for each cDNA or gDNA target containing a R2N overhang.
  • second droplet generation where the first droplet is fused with a cell barcoding bead, PCR reagents and a forward primer for each target that contains a CS overhang.
  • Tm conditions of the multiplexed PCR favor binding of the primers to its cDNA and gDNA targets for the first 10 PCR cycles.
  • Tm conditions for the second 10 PCR cycles favor binding of those PCR products to the CS barcoding overhangs introducing a unique CBC within each separate droplet.
  • the different fixing conditions could be discriminated by using two distinct SBCs for each condition in the in-situ RT reaction (Fig. 1). Analysis was performed using UMI-tools (6). [0161] An initial ranking of cells per reads yielded 24,468 cells that lie above an automatically determined threshold (Fig.2B). After filtering out low quality cells (less than 10 cDNA/gDNA targets and less than 80% of reads associated with a given SBC) we obtained 9,364 cells that are evenly distributed over both fixation conditions (Fig. 2C). The type of fixation did not have an impact for gDNA target amplification (Fig.2D-F). However, glyoxal fixation yielded higher cDNA targets/cell and UMIs/cell than PFA (Fig.
  • cDNA targets showed varying levels of expression while some were expressed only in a fraction of the cells (Fig. 3B, G and H).
  • Housekeeping genes like GAPDH and TUBB2B were expressed in all cells, in addition to genes like POU5F1, SOX2 and STAT3 that are crucial for iPSC maintenance.
  • Comparison of gene expression levels to bulk RNA-seq data of summed up cDNA UMIs or reads of all cells showed comparable expression levels for the vast majority of targets (Fig. 3C).
  • Unbiased clustering based on the expressed cDNA targets revealed several clusters (Fig. 3D), while clusters were mainly driven by genes that were expressed only in a subpopulation of cells.
  • gRNAs using a CRISPRi prediction tool (9, 10) to perturb the transcription start site of all the predicted genes affected by the eQTLs (CRISPRi controls) and non-targeting control (NTC) gRNAs that should not show any effect.
  • CRISPRi controls CRISPRi controls
  • NTC non-targeting control
  • the lentiviral CROP-seq gRNA library was used to infect cells that constitutively express a CRISPRi transgene from the AAVS1 locus in iPSCs (Fig. 4C). Infected cells were selected for by flow cytometry and the scDNA-scRNA-seq method was performed as described above. gRNAs were confidently assigned to cells with a mean coverage of 46 cells/gRNA (Fig. 4D) and tested if the cause any expression changes within the cells.
  • NTC gRNAs did not show any effect on any of the expressed genes, while the 20 out of 21 CRISPRi controls (95 %) showed a significant down-regulation of the intended target gene.7 out of 30 predicted eQTLs (23 %) showed an effect on their predicted target gene. This highlights the power of the method to detect expression changes in a quantitative manner.
  • Fig.5A we aimed to directly install eQTLs and read out associated gene expression changes. For this we screened the 29 eQTLs from the previous CRISPRi perturbation screen while including 5 coding controls that install STOP codons in the beginning of the targeted transcript. We expected to observe non-sense mediated decay in these controls.
  • Transgenic iPSCs that expressed an improved version of the prime editor (PEmax) or PEmax together with MLH1dn (11) were lipofected with a construct expressing pegRNAs that aim to introduce the specific edits (Fig.5B). Lipofected cells were selected for by flow cytometry and scDNA-scRNA-seq was performed. Cells were assigned to be either homozygous- (E+/E+), heterozygous- (E+/E-) and non-edited (E-/E-) depending on the fraction of edited reads found for a given target. Overall, we observed limited editing efficiency with both PEmax cell lines tested (Fig. 5C).
  • This single-cell multiomic readout is crucial to precisely link genotype with gene expression profiles due to differential editing efficiencies at distinct loci.
  • Other applications include to characterize patient samples for mutational status and associated gene expression. This could lead to better predictions for treatment of cancer patients, while it will drive mechanistic insights of disease-relevant eQTL mappings.
  • it enables to perform lineage tracing analysis using endogenous gDNA or mtDNA loci. Due to high mutational rates observed in mtDNA this could already enable lineage dissection of patient samples without any further modifications of the described method.
  • Example 3
  • This example illustrates the methods of developing targeted droplet-based scDNA- scRNA-seq (SDR-seq) that is amenable to screen genetic variation in high-throughput.
  • SDR-seq targeted droplet-based scDNA- scRNA-seq
  • SDR-seq droplet-based multiomic targeted scDNA-scRNAseq
  • Genomic variation in both coding and non-coding regions of the genome is influencing human population differences and driving diseases in a mono-genetic or complex manner 1–3 .
  • Genetic loss-of-function screening of coding genes and CRISPRi/CRISPRa screens in non-coding regions have contributed valuable insights into disease mechanisms but lacks taking into account information about precise genomic variation 4–6 . This potentially masks more complex cellular disease phenotypes that are caused by individual variants in a gene or in non-coding regions 7 .
  • Existing precision genome editing tools have limited efficiency and variable editing outcome in mammalian cells hindering a systematic study of genetic variation and its impact on disease-relevant gene expression 8–10 .
  • RNA and gDNA To confidently link a precise genotype to gene expression a combined single-cell RNA and gDNA is required.
  • Current technologies for simultaneous high-sensitivity readout of both RNA and gDNA are well-based and laborious with limited throughput 11–14 .
  • High-throughput droplet-based or split-pooling approaches enabling to measure thousands of cells simultaneously are currently possible for a multitude of readouts but are lacking for a combined high-sensitivity and tagmentation-independent readout of RNA and gDNA 15–17 .
  • SDR- seq targeted droplet-based scDNA-scRNA-seq
  • SDR-seq Droplet-based scDNA-scRNA-seq
  • a first droplet generation cells are lysed, treated with proteinase K and mixed with reverse primer for each intended gDNA or cDNA target. This is followed by a second droplet generation where the first droplet is fused with a cell barcoding bead, PCR reagents and a forward primer for each target with a CS overhang.
  • a multiplexed PCR both gDNA and cDNA targets are amplified and cell barcoding occurs by complementary CS overhangs of the PCR amplicons and oligos from the cell barcoding bead. Emulsions are broken after the PCR and sequencing ready libraries generated.
  • RNA targets Coverage for RNA targets is expected to be varying, as not all genes are expressed in all cells and to the same degree and targets were chosen based on a range of expression values to check the sensitivity of the method.23 out of 28 gDNA targets (82 %) showed high coverage and were found in almost all the cells sequenced (Figs. 7G-I). Little differences were observed in gDNA target detection and coverage using either PFA or glyoxal (Figs.11E, G). As expected, individual RNA targets showed varying levels of expression while some were expressed only in a fraction of the cells (Figs.7J-L). Both RNA targets detected and UMI coverage were increased when using glyoxal compared to the PFA for fixation (Figs. 8F, H).
  • a highly sensitive gene expression readout is crucial due to the potential limited impact of individual variants on gene expression.
  • Ubiquitously expressed housekeeping or iPSC-maintenance genes were detected in all cells, while other genes showed specific expression only in a subset of cells (Fig.9).
  • Comparison of gene expression levels to bulk RNA-seq data of summed up UMIs of all cells showed comparable expression levels for the vast majority of targets with high correlation (Figs. 7M, N). This shows that SDR-seq is capable of a highly sensitive readout of a multitude of DNA and RNA targets in thousands of single cells in a single experiment with the potential to link those modalities in a high-throughput fashion.
  • SDR-seq is scalable to detect hundreds of targets with high confidence [0173] Different sequencing modalities in terms of sequencing length and depth would be preferred for either gDNA or RNA targets. For gDNA targets the entire amplicon should be sequenced to ensure that each possible variant is covered. On the contrary, for RNA targets only the barcode structure (cell BC, sample BC and UMI) and the transcript information is required while adjusting the sequencing depth would allow for higher sensitivity to detect potential gene expression changes. To enable this, we modified the overhangs of the reverse primers to contain either R2N (gDNA) or R2 (RNA) overhangs allowing to generate separate NGS libraries (Fig. 11A).
  • a gDNA target was counted as detected per cell if it was covered with more than 5 reads per cell, while an RNA target was counted if it was detected by 1 UMI per cell.
  • Fig.12A-C Detection and coverage of shared gDNA targets between panels was well correlated indicating reproducible detection of gDNA targets independent of the panel size (Figs. 10B, C).
  • the minor decrease in detection rate for the bigger panel sizes predominantly affected targets with lower coverage across panels (Figs. 12D, E, Fig. 13A).
  • RNA targets we observed that there was as only a minor decrease in targets detected in larger panels compared to the 120 panel (Extended Data Fig.5F-H). Detection and gene expression of shared RNA targets were highly correlated between all panels (Fig. 10D, E, Figs. 12I, J) showing a robust and sensitive detection of gene expression readouts. Similar to gDNA targets, variability was predominantly observed for lowly expressed genes (Fig.13B). [0175] To check whether chromosomal context has an influence on detection we included targets sites that were either overlapping (OEG) or not overlapping expressed genes (NOEG), and were tested for different chromatin marks indicating different regulatory elements depending on their proximity to the TSS (Fig 10E) 21 .
  • OEG overlapping
  • NOEG not overlapping expressed genes
  • SDR-seq is scalable to assay hundreds of gDNA and RNA targets simultaneously with high reproducibility and sensitivity across different panel sizes. This makes it a versatile tool to both assay variants in a multitude of loci in single cells, while linked gene expression changes and cell identity markers can be measured. SDR-seq is sensitive to confidently detect gene expression changes [0178] Genomic variants might increase or decrease gene expression levels of distinct genes, while effect sizes are expected to be small. Therefore, it is critical to have a highly sensitive gene expression readout that can confidently detect changes.
  • NTC non- targeting control gRNAs
  • TSS transcription start site
  • Fig. 14A gRNAs that target the gene body of a transcript where a possible STOP codon could be introduced via prime editing
  • iPSCs expressing the CRISPRi transgene from the safe-harbor locus AAVS1 were infected with the lentiviral CROP-seq gRNA library, selected via FACS and SDR-seq was performed (Fig. 14B).
  • Cells were assigned to gRNAs (75%) with an average coverage of 30 cells/gRNA (Figs. 15A, B).
  • NTC gRNAs did not show a significant effect on any of the genes that were measured, while the vast majority of CRISPRi control gRNAs targeting the TSS of a gene showed a strong effect on the expression levels of the associated target gene (Fig. 14C). 7 eQTL gRNAs and 3 STOP control gRNAs showed a significant effect on target gene expression.
  • PE prime editing
  • PEmax or PEmax-MLH1dn a dominant negative regulator of the mismatch repair pathway that was shown to increase editing efficiency
  • Fig. 15D a fluorescent lentiviral reporter system that enables to measure editing efficiencies via the reconstitution of a non-functional EGFP
  • pegRNA prime editing gRNA
  • Fig. 15E a prime editing gRNA
  • Genotypes could be confidently called discriminating between reference (REF), heterozygous (HET) and alternative (ALT) alleles (Figs. 16C-E).
  • REF reference
  • HAT heterozygous
  • ALT alternative
  • Figs. 16C-E alternative (ALT) alleles
  • Figs. 16C-E effects of nonsense mediated decay on transcript levels can vary 23 .
  • STOP codons introduced in ATF4 and MYH10 had a significant impact on gene expression (Fig.14G).
  • Fig.14G As the PE iPSCs exhibited only limited editing efficiency we aimed to install eQTLs via base editing in a follow up experiment. We selected 56 high confidence eQTLs to be installed with either ABE8e or CBE base editors (Fig.14H).
  • Fig.14I After introducing gRNA libraries into iPSCs, cells were selected and SDR-seq performed (Fig.14I). In this experiment we found several eQTL variants having a significant effect on target gene expression (Fig. 14J). In particular a synonymous variant in POU5F1 showed a significant effect on gene expression (Fig. 17A). However, after assessing variants along the entire amplicon of POU5F1 we found a diverse set of variants that had an impact on gene expression (Fig. 14K, Fig. 17B). In particular variants in the 3’ UTR were associated with differential transcript levels. This highlights the importance of directly assessing variants at the locus of interest to get a better resolution on the impact on gene expression.
  • E8+RI 10 ⁇ M Y-27632 dihydrochloride
  • 1.5 x10 ⁇ 6 cells were transferred to a new 15 ml conical tube and spun at 500 g for 3 min.
  • wash buffer 1 1 ml ice-cold wash buffer 1 (1x PBS with 2% BSA, 1 mM DTT and 0.5 U/ ⁇ l RNasin® Plus Ribonuclease Inhibitor - Promega #N2615) was added and cells were spun at 500 g for 3 min at 4 C. Supernatant was carefully removed and wash step was repeated with wash buffer 1 for a total of 2 washes.
  • Cells were resuspended in 175 ⁇ l ice-cold permeabilization buffer (10 mM TRIS- HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.2 U/ ⁇ l RNasin® Plus Ribonuclease Inhibitor, 1mM DTT, 2 % BSA, 0.1 % IGEPAL CA-630 and 0.01 % Digitonin) and incubated for 4 min on ice.
  • ice-cold permeabilization buffer 10 mM TRIS- HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.2 U/ ⁇ l RNasin® Plus Ribonuclease Inhibitor, 1mM DTT, 2 % BSA, 0.1 % IGEPAL CA-630 and 0.01 % Digitonin
  • Cells were spun at 500 g for 3 min at 4 °C, supernatant removed, resuspended in 300 ⁇ l of 0.5x PBS with 0.2 U/ ⁇ l RNasin® Plus Ribonuclease Inhibitor, filtered through a 40 ⁇ m strainer, counted and diluted to 1.4 x10 ⁇ 6 cells/ml.
  • RT master mix consisting of a final concentration of 1x RT Buffer, 0.25 U/ ⁇ l Enzymatics RNAse Inhibitor (Biozym – 180520), 0.2 U/ ⁇ l RNasin® Plus Ribonuclease Inhibitor, 500 mM dNTPs and 20 U/ ⁇ l Maxima H Minus Reverse Transcriptase (ThermoFisher - EP0752) was prepared on ice for in 8 ⁇ l for a total reaction volume of 20 ⁇ l. 4 ⁇ l of reverse transcription oligo (12.5 ⁇ M) were combined in each 96-well plate with 8 ⁇ l reverse transcription master mix.
  • RT 8 ⁇ l fixed and permeabilized cells (10000 cells total) were added to each well, yielding a total reaction volume of 20 ⁇ l.
  • Reverse transcription was performed in a thermocycler using the protocol shown in Table 12. All RT reactions were pooled into a 15 ml conical tube, 1 volume of ice-cold 1x PBS with 1% BSA was added and cells were spun at 500 g for 5 min and supernatant was removed. Table 12.
  • Reverse transcription (RT) protocol [0189] Samples were processed using the Tapestri microfluidic device from Mission Bio (MB51-0007, MB51-0010, MB51-0009) according to manufactures protocol with modifications.
  • In-situ RT processed cell pellet from previous step was resuspended in the cell buffer of Mission Bio, cells were counted and diluted to the appropriate concentration of 3000- 4000 cells/ ⁇ l.
  • Custom primers were used in the multiplexed droplet PCR amplification step.
  • cDNA primers were designed using the TAP-seq primer prediction tool with at targeted Tm of 60°C 4 .
  • gDNA primers were designed using the Tapestri Designer (https://designer.missionbio.com). Version 1 gDNA and cDNA primers both had CS and R2N overhangs (only used in proof-of-concept experiment). Version 2 gDNA primers had CS and R2N, whereas cDNA primers had CS and R2 overhangs.
  • Knipping P. Ravisankar, P.-F. Chen, C. Chen, J. W. Nelson, G. A. Newby, M. Sahin, M. J. Osborn, J. S. Weissman, B. Adamson, D. R. Liu, Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell.184, 5635-5652.e29 (2021). 12. A. Buniello, J. A. L. MacArthur, M. Cerezo, L. W. Harris, J. Hayhurst, C. Malangone, A. McMahon, J. Morales, E. Mountjoy, E. Sollis, D. Suveges, O. Vrousgou, P. L. Whetzel, R.
  • a method for simultaneously detecting RNA and genomic DNA (gDNA) within the same cell comprising: (i) providing a single cell suspension comprising fixed and permeabilized cells; (ii) performing an in-situ reverse transcription (RT) step to generate cDNA molecules by contacting the cells with reverse transcriptase and a RT primer; (iii) lysing the cells in a first droplet comprising a first reverse primer that hybridizes to the cDNA molecules and a second reverse primer that hybridizes to the gDNA molecules, wherein the first or second reverse primer comprises a R2N or R2 overhang sequence; (iv) fusing the first droplet to a second droplet comprising PCR reagents and a forward primer that hybridizes to both the cDNA and gDNA molecules and comprises a capture sequence (CS) overhang sequence; and (v) simultaneously amplifying the cDNA and gDNA molecules, thereby detecting both RNA and gDNA within the same cell.
  • RT in-situ
  • Embodiment 2 The method of embodiment 1, wherein the single cell suspension is fixed with paraformaldehyde (PFA) or glyoxal.
  • Embodiment 3 The method of embodiment 1 or 2, wherein the R2N overhang sequence comprises the nucleic acid sequence GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG (SEQ ID NO:2), and the R2 overhang sequence comprises the nucleic acid sequence GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO:3).
  • the RT primer comprises a capture sequence (CS), a sample barcode (SBC) sequence, a unique molecular identifier (UMI) sequence, and a sequence that binds to mRNA.
  • CS capture sequence
  • SBC sample barcode
  • UMI unique molecular identifier
  • Embodiment 5 The method of embodiment 4, wherein the CS sequence hybridizes to a complementary sequence attached to a solid support, the SBC sequence comprises a known sequence of variable length, and the UMI sequence comprises a random sequence of variable length.
  • Embodiment 6. The method of any one of embodiments 1 to 5, wherein the CS comprises the nucleic acid sequence GTACTCGCAGTAGTC (SEQ ID NO:1).
  • Embodiment 8 The method of embodiment 7, wherein the SBC sequence comprises a nucleic acid sequence selected from the group consisting of TCGCCTTA (SEQ ID NO:4), CTAGTACG (SEQ ID NO:5), TTCTGCCT (SEQ ID NO:6), GCTCAGGA (SEQ ID NO:7), AGGAGTCC (SEQ ID NO:8), CATGCCTA (SEQ ID NO:9), GTAGAGAG (SEQ ID NO:10), and CCTCTCTG (SEQ ID NO:11).
  • TCGCCTTA SEQ ID NO:4
  • CTAGTACG SEQ ID NO:5
  • TTCTGCCT SEQ ID NO:6
  • GCTCAGGA SEQ ID NO:7
  • AGGAGTCC SEQ ID NO:8
  • CATGCCTA SEQ ID NO:9
  • GTAGAGAG SEQ ID NO:10
  • CCTCTCTG SEQ ID NO:11
  • Embodiment 10 The method of any one of embodiments 4 to 8, wherein the UMI sequence is from 4 to 50 nucleotides in length.
  • Embodiment 10 The method of embodiment 9, wherein the UMI sequence comprises the nucleic acid sequence NNNNNN (SEQ ID NO:12).
  • Embodiment 11 The method of embodiment 4, wherein the sequence that binds to mRNA comprises oligo(dT) or a sequence that hybridizes to any region of the mRNA.
  • step (v) comprises PCR conditions that favor binding of forward and reverse primers to the cDNA and gDNA molecules thereby producing a first set of PCR products each comprising the CS.
  • Embodiment 13 The method of embodiment 12, wherein the second droplet further comprises cell barcode oligonucleotides each comprising a cell barcode (CBC) and a sequence complementary to the CS, thereby the first set of PCR products hybridize to the cell barcode oligonucleotides, producing a second set of PCR products each comprising the CS and the CBC.
  • Embodiment 14 The method of embodiment 13, wherein the cell barcode oligonucleotides are attached to a solid support in the second droplet prior step (iv) and released from the solid support after the second droplet is fused with the first droplet.
  • Embodiment 16 The method of any one of embodiments 1 to 14, wherein simultaneous amplification in step (v) produces a cDNA library and a gDNA library.
  • Embodiment 16 The method of embodiment 15, further comprising sequencing the cDNA and gDNA libraries.
  • Embodiment 17. The method of embodiment 16, wherein the cDNA library is sequenced by a first sequencing modality, and the gDNA library is sequenced by a second sequencing modality.
  • Embodiment 18 The method of embodiment 17, wherein the first and second sequencing modalities are the same or different.
  • Embodiment 19 The method of embodiment 17 or 18, wherein the first sequencing modality comprises scRNA-seq and the second sequencing modality comprises scDNA-seq.
  • Embodiment 20 The method of embodiment 17 or 18, wherein the first sequencing modality comprises Illumina® next generation sequencing (NGS), and the second sequencing modality comprises Nextera® NGS, or the first sequencing modality comprises Nextera® NGS, and the second sequencing modality comprises Illumina® NGS.
  • Embodiment 21 The method of any one of embodiments 1 to 20, wherein step (iii) comprises contacting the cells with proteinase K to lyse the cells.
  • Embodiment 22 The method of any one of embodiments 1 to 21, wherein the cell is a prokaryotic or eukaryotic cell.
  • Embodiment 23 Embodiment 23.
  • Embodiment 24 The method of any one of embodiments 1 to 22, wherein the cell is an induced pluripotent stem cell (iPSC).
  • iPSC induced pluripotent stem cell
  • Embodiment 24 The method of any one of embodiments 1 to 23, wherein the cell is a genetically modified cell.
  • Embodiment 25 The method of embodiment 24, wherein the cell is modified using a CRISPR/Cas gene editing system.
  • Embodiment 26. The method of embodiment 25, wherein the CRISPR/Cas gene editing system is CRISPR interference (CRISPRi).
  • Embodiment 27 Embodiment 27.
  • RNA expressed by the genetically modified cell is sequenced by scRNA-seq and genomic DNA is sequenced by scDNA-seq.
  • Embodiment 28 A reaction mixture comprising RNA and gDNA from a single cell, a reverse transcriptase, and a RT primer comprising a capture sequence (CS), a sample barcode (SBC) sequence, a unique molecular identifier (UMI) sequence, and a sequence that binds to mRNA.
  • CS capture sequence
  • SBC sample barcode
  • UMI unique molecular identifier
  • Embodiment 30 The reaction mixture of embodiment 28 or 29, wherein the CS comprises the nucleic acid sequence GTACTCGCAGTAGTC (SEQ ID NO:1).
  • Embodiment 31 The reaction mixture of any one of embodiments 28 to 30, wherein the SBC sequence is from 4 to 50 nucleotides in length.
  • Embodiment 32 Embodiment 32.
  • TCGCCTTA SEQ ID NO:4
  • CTAGTACG SEQ ID NO:5
  • TTCTGCCT SEQ ID NO:6
  • GCTCAGGA SEQ ID NO:7
  • AGGAGTCC SEQ ID NO:8
  • CATGCCTA SEQ ID NO:9
  • GTAGAGAG SEQ ID NO:10
  • CCTCTCTG SEQ ID NO:11
  • Embodiment 35 The reaction mixture of any one of embodiments 28 to 34, wherein the UMI sequence comprises the nucleic acid sequence NNNNNNNN (SEQ ID NO:12).
  • Embodiment 35 The reaction mixture of any one of embodiments 28 to 34, further comprising cDNA molecules produced by reverse transcription of the RNA and a reverse primer that hybridizes to both the cDNA and the gDNA molecules and comprises a R2N or R2 overhang sequence.
  • Embodiment 36 Embodiment 36.
  • the reaction mixture of embodiment 35, wherein the reverse primer comprising the R2N overhang sequence comprises the nucleic acid sequence GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG (SEQ ID NO:2) and the reverse primer comprising the R2 overhang sequence comprises the nucleic acid sequence GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO:3).
  • Embodiment 37 The reaction mixture of any one of embodiments 28 to 36, comprising a, one or more, or a plurality of polynucleotides selected from a sequence in any one of SEQ ID Nos: 1-83.
  • Embodiment 38 A polynucleotide comprising a capture sequence (CS).
  • Embodiment 39 The polynucleotide of embodiment 38, further comprising a sample barcode (SBC) sequence, a unique molecular identifier (UMI) sequence, and/or a sequence that binds to mRNA.
  • SBC sample barcode
  • UMI unique molecular identifier
  • Embodiment 40 The polynucleotide of embodiment 39, wherein the SBC sequence comprises a known sequence of variable length, and the UMI sequence comprises a random sequence of variable length.
  • Embodiment 41 The polynucleotide of any one of embodiments 38 to 40, wherein the CS comprises the nucleic acid sequence GTACTCGCAGTAGTC (SEQ ID NO:1).
  • Embodiment 42 The polynucleotide of any one of embodiments 38 to 40, wherein the CS comprises the nucleic acid sequence GTACTCGCAGTAGTC (SEQ ID NO:1).
  • Embodiment 43 The polynucleotide of any one of embodiments 39 to 42, wherein the SBC sequence comprises a nucleic acid sequence selected from the group consisting of TCGCCTTA (SEQ ID NO:4), CTAGTACG (SEQ ID NO:5), TTCTGCCT (SEQ ID NO:6), GCTCAGGA (SEQ ID NO:7), AGGAGTCC (SEQ ID NO:8), CATGCCTA (SEQ ID NO:9), GTAGAGAG (SEQ ID NO:10), and CCTCTCTG (SEQ ID NO:11).
  • TCGCCTTA SEQ ID NO:4
  • CTAGTACG SEQ ID NO:5
  • TTCTGCCT SEQ ID NO:6
  • GCTCAGGA SEQ ID NO:7
  • AGGAGTCC SEQ ID NO:8
  • CATGCCTA SEQ ID NO:9
  • GTAGAGAG SEQ ID NO:10
  • CCTCTCTG SEQ ID NO:11
  • Embodiment 44 The polynucleotide of any one of embodiments 39 to 43, wherein the UMI sequence is from 4 to 50 nucleotides in length.
  • Embodiment 45 The polynucleotide of any one of embodiments 39 to 44, wherein the UMI comprises the nucleic acid sequence NNNNNNNN (SEQ ID NO:12).
  • Embodiment 46 The polynucleotide of any one of embodiments 39 to 45, wherein the sequence that binds to mRNA comprises oligo(dT) or a sequence that hybridizes to any region of the mRNA.
  • Embodiment 47 Embodiment 47.

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Abstract

La présente divulgation concerne un procédé de mesure de l'ARN et de l'ADNg simultanément dans la même cellule d'une manière ciblée avec une couverture élevée dans toutes les cellules. La méthode s'effectue par combinaison d'une transcription inverse (RT) in situ avec une PCR multiplexée dans des gouttelettes.
PCT/US2024/029950 2023-05-19 2024-05-17 Lecture multiomique à haut débit d'arn et d'adn génomique dans des cellules uniques Pending WO2024243040A2 (fr)

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US20180080021A1 (en) * 2016-09-17 2018-03-22 The Board Of Trustees Of The Leland Stanford Junior University Simultaneous sequencing of rna and dna from the same sample
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