WO2025199320A1 - Procédés et compositions pour caractériser le transcriptome - Google Patents

Procédés et compositions pour caractériser le transcriptome

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Publication number
WO2025199320A1
WO2025199320A1 PCT/US2025/020706 US2025020706W WO2025199320A1 WO 2025199320 A1 WO2025199320 A1 WO 2025199320A1 US 2025020706 W US2025020706 W US 2025020706W WO 2025199320 A1 WO2025199320 A1 WO 2025199320A1
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Prior art keywords
reverse transcriptase
sequence
mutation
engineered
sample
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Dan LANDAU
Ivan Raimondi
Husain DANISH
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Cornell University
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Cornell University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07049—RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase

Definitions

  • This disclosure generally relates to the field of transcriptomics, such as single-cell and spatial transcriptomics.
  • RNA sequence information is essential for spatially evaluating gene expression, including evaluating how sequence variations affect cellular phenotypes.
  • Single-cell and spatial transcriptomics techniques traditionally use contiguous probe pairs that hybridize to RNA target sequence and are then sequenced to obtain RNA sequence information. However, such techniques do not capture RNA sequence variations such as mutations, deletions, or duplications in the target RNA transcript. Therefore, compositions and methods useful for leveraging the advantages of single-cell spatial transcriptomics and accurate retrieval of RNA transcript sequences are needed.
  • RNA transcripts Methods and compositions that leverage single-cell and spatial transcriptomics to identify single nucleotide variations, small deletions and/or insertions in RNA transcripts are needed. Methodologies to achieve this are described herein. In particular, described herein are new and improved methods to genotype the transcriptome and/or profile or identify single nucleotide variations, small deletions and/or insertions. The methods described herein can be used in fixed samples (e.g., formalin-fixed samples).
  • an engineered reverse transcriptase wherein a wildtype reverse transcriptase is engineered to comprise: a) a first mutation that inhibits, reduces, interferes with, substantially abolishes, or abolishes the strand displacement activity of the reverse transcriptase relative to the wild-type reverse transcriptase, wherein the first mutation is one or more substitutions, deletions or insertions of amino acids (e.g., wherein the first mutation is an amino acid substitution such as any one or more described herein); and/or b) a second mutation that inhibits, reduces, interferes with, substantially abolishes, or abolishes RNAse-H activity of the reverse transcriptase relative to the wild-type reverse transcriptase, wherein the second mutation is one or more substitutions, deletions or insertions of amino acids (e.g., wherein the second mutation is an amino acid substitution such as any one or more described herein).
  • the inhibiting or reducing enzymatic activity is inhibiting or reducing enzymatic activity by at least or more than 25%, 30%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.
  • the reverse transcriptase strand displacement activity is measured by any means known in the art or disclosed herein.
  • reverse transcriptase RNAse-H activity is measured by any means known in the art or disclosed herein.
  • one or both of these activities are inhibited by at least or more than 80%, 85%, 90%, 95%, 97%, 98%, or 99%, or to the level such that the activity is no longer detectable by one or more assays of such activity known in the art or described herein.
  • an engineered reverse transcriptase wherein a wildtype reverse transcriptase is engineered to comprise: a) a first mutation that inhibits, reduces, interferes with, substantially abolishes, or abolishes the strand displacement activity of the reverse transcriptase relative to the wild-type reverse transcriptase, wherein the first mutation is one or more substitutions, deletions or insertions of amino acids (e.g., wherein the first mutation is an amino acid substitution such as any one or more described herein); and/or b) a second mutation that inhibits, reduces, interferes with, substantially abolishes, or abolishes digestion of DNA:RNA hybrid by the reverse transcriptase relative to the wild-type reverse transcriptase, wherein the second mutation is one or more substitutions, deletions or insertions of amino acids (e.g., wherein the second mutation is an amino acid substitution such as any one or more described herein).
  • the inhibiting or reducing enzymatic activity is inhibiting or reducing enzymatic activity by at least or more than 25%, 30%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.
  • the reverse transcriptase strand displacement activity is measured by any means known in the art or disclosed herein.
  • the digestion of DNA:RNA hybrid by reverse transcriptase is measured by any means known in the art or disclosed herein.
  • one or both of these activities are inhibited by at least or more than 80%, 85%, 90%, 95%, 97%, 98%, or 99%, or to the level such that the activity is no longer detectable by one or more assays of such activity known in the art or described herein.
  • the wild-type reverse transcriptase is any natural reverse transcriptase.
  • the wild-type reverse transcriptase is HIV-1 reverse transcriptase, M-MLV reverse transcriptase, AMV reverse transcriptase, or Telomerase reverse transcriptase.
  • the wild-type reverse transcriptase is Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) and/or comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1.
  • the engineered reverse transcriptase comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% identical to SEQ ID NO: 2 and further comprises a methionine (M) added at the beginning of the sequence (as the first amino acid).
  • M methionine
  • the first mutation is in the first finger domain of the wild-type reverse transcriptase.
  • the first finger domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3.
  • the second mutation is in the RNAse-H domain of the wild-type reverse transcriptase.
  • the RNAse-H domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 4.
  • the first mutation is in or corresponds to amino acid Y64 (e.g., Y64A) relative to the amino acid sequence numbering of Moloney Murine Leukemia Virus Reverse Transcriptase or SEQ ID NO: 1, and optionally wherein the engineered reverse transcriptase is at least 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1 or comprises a sequence that is at least 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3.
  • the first mutation is or corresponds to Y64A relative to the amino acid sequence numbering of Moloney Murine Leukemia Virus Reverse Transcriptase or SEQ ID NO: 1.
  • the engineered reverse transcriptase comprises a sequence identical to SEQ ID NO: 1 or SEQ ID NO: 3 except for the Y64 (e.g., Y64A) mutation.
  • the second mutation is in or corresponds to amino acid D524, D583 or E562 (e.g., D524A, D524G, D583N, D524N, or E562Q) relative to the amino acid sequence numbering of Moloney Murine Leukemia Virus Reverse Transcriptase or SEQ ID NO: 1, and optionally wherein the engineered reverse transcriptase is at least 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1 or comprises a sequence that is at least 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 4.
  • D524, D583 or E562 e.g., D524A, D524G, D583N, D524N, or E562Q
  • the engineered reverse transcriptase is at least 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1 or comprises a sequence that is at least
  • the second mutation is or corresponds to D524A, D524G, D583N, D524N, or E562Q relative to the amino acid sequence numbering of Moloney Murine Leukemia Virus Reverse Transcriptase or SEQ ID NO: 1.
  • the engineered reverse transcriptase comprises a sequence identical to SEQ ID NO: 1 or SEQ ID NO: 4 except for the D524, D583 or E562 (e.g., D524A, D524G, D583N, D524N, or E562Q) mutation.
  • the first mutation is in or corresponds to amino acid Y64 or Y64A of M-MLV RT
  • the second mutation is in or corresponds to amino acid D524, D583 or E562, or D524A, D524G, D583N, D524N, or E562Q, of M-MLV RT.
  • the first mutation is or corresponds to Y64A (tyrosine-to-alanine at position 64) and/or the second mutation is or corresponds to D524A (aspartic acid-to-alanine at position 524) relative to the amino acid sequence numbering of Moloney Murine Leukemia Virus Reverse Transcriptase or SEQ ID NO: 1, optionally wherein the engineered reverse transcriptase is at least 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1 or comprises a sequence that is at least 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3 and/or SEQ ID NO: 4.
  • the engineered reverse transcriptase has a sequence identical to SEQ ID NO: 1 except for an Y64 (e.g., Y64A) mutation and an D524, D583 or E562 (e.g., D524A, D524G, D583N, D524N, or E562Q) mutation.
  • Y64 e.g., Y64A
  • D524, D583 or E562 e.g., D524A, D524G, D583N, D524N, or E562Q
  • the engineered reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the engineered reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 2 and further comprises a methionine (M) added at the beginning of the sequence (as the first amino acid). In some embodiments, and without being bound by any theory, M is added at the beginning of the sequence of reverse transcriptase to improve or facilitate recombinant protein engineering and/or production.
  • M methionine
  • the engineered reverse transcriptase comprises an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2, wherein the sequence comprises Y64 (e.g., Y64A) substitution and an D524, D583 or E562 (e.g., D524A, D524G, D583N, D524N, or E562Q) substitution, and optionally wherein the sequence further comprises one or more amino acids at the beginning of the sequence, which amino acids may facilitate protein production (e.g., wherein the sequence further comprises M as the first amino acid at the beginning of the sequence).
  • Y64 e.g., Y64A
  • D524, D583 or E562 e.g., D524A, D524G, D583N, D524N, or E562Q
  • the engineered reverse transcriptase comprises the first finger domain having the amino acid sequence of SEQ ID NO: 7. In some embodiments, the engineered reverse transcriptase comprises the RNAse-H domain having the amino acid sequence of SEQ ID NO: 8. In some embodiments, the engineered reverse transcriptase comprises the amino acid sequence of SEQ ID NO: 7 and/or the amino acid sequence of SEQ ID NO: 8.
  • this disclosure provides a composition comprising the engineered reverse transcriptase disclosed herein. In some aspects, this disclosure provides a kit comprising the engineered reverse transcriptase disclosed herein.
  • this disclosure provides a nucleic acid encoding the engineered reverse transcriptase disclosed herein.
  • RNA ribonucleic acid sequence
  • a method for analyzing a target ribonucleic acid sequence (RNA) in a sample comprising a) contacting the sample with a first polynucleotide probe comprising a nucleotide sequence substantially complementary to a first sequence of the target RNA and a second polynucleotide probe comprising a nucleotide sequence substantially complementary to a second sequence of the target RNA, wherein the first sequence and the second sequence are separated by a third sequence of the target RNA comprising at least one nucleotide; b) contacting the sample with the engineered reverse transcriptase described herein (such as any of the engineered reverse transcriptases described herein), whereby the first polynucleotide probe is extended to produce a DNA complementary to the third sequence (cDNA); and c) conducting a ligation reaction to ligate the cDNA to the second polynucleotide probe (e.g., contacting the sample with a liga
  • the third sequence comprises up to 100 nucleotides, up to 75 nucleotides, up to 50 nucleotides, up to 40 nucleotides, up to 30 nucleotides, up to 25 nucleotides, or up to 20 nucleotides. In some embodiments, the third sequence comprises about 1 to 100 nucleotides. In some embodiments, the third sequence comprises about 1 to 75 nucleotides. In some embodiments, the third sequence comprises about 1 to 50, 1 to 40, 1 to 30, or 1 to 20 nucleotides. In some embodiments, the third sequence comprises about 2 to 100 nucleotides. In some embodiments, the third sequence comprises about 2 to 75 nucleotides.
  • the third sequence comprises about 3 to 40 nucleotides. In some embodiments, the third sequence comprises about 4 to 100 nucleotides. In some embodiments, the third sequence comprises about 4 to 75 nucleotides. In some embodiments, the third sequence comprises 4 to 50, 4 to 40, 4 to 35, 4 to 30, 4 to 25, or 4 to 20 nucleotides. In some embodiments, the third sequence comprises about 5 to 100 or 6 to 100 nucleotides. In some embodiments, the third sequence comprises about 5 to 75 or 6 to 75 nucleotides. In some embodiments, the third sequence comprises 5 to 50, 5 to 40, 5 to 35, 5 to 30, 5 to 25, or 5 to 20 nucleotides.
  • the third sequence comprises 6 to 50, 6 to 40, 6 to 35, 6 to 30, or 6 to 25 nucleotides. In some embodiments, the third sequence comprises 6 to 20 nucleotides. In some embodiments, the third sequence comprises 6 to 15, 6 to 12, 6 to 10 nucleotides. In some embodiments, the third sequence comprises 7 to 50, 7 to 40, 7 to 35, 7 to 30, 7 to 25, 7 to 20, 7 to 15, 7 to 12, or 7 to 10 nucleotides. In some embodiments, the third sequence comprises 8 to 50, 8 to 40, 8 to 35, 8 to 30, 8 to 25, 8 to 20, or 8 to 15 nucleotides.
  • the third sequence comprises 9 to 50, 9 to 40, 9 to 35, 9 to 30, 9 to 25, 9 to 20, or 9 to 15 nucleotides. In some embodiments, the third sequence comprises 9 to 20 nucleotides. In some embodiments, the target RNA is mRNA.
  • the method provided herein utilizes an engineered reverse transcriptase (e.g., from M-MLV) in which the strand displacement activity is inhibited, reduced, interfered with, substantially abolished, or abolished as described herein, and optionally wherein the RNAse-H activity is inhibited, reduced, interfered with, substantially abolished, or abolished as described herein (e.g., comprising the sequence of SEQ ID NO: 2, or a variant thereof in which such activity or activities are inhibited).
  • an engineered reverse transcriptase e.g., from M-MLV
  • the RNAse-H activity is inhibited, reduced, interfered with, substantially abolished, or abolished as described herein (e.g., comprising the sequence of SEQ ID NO: 2, or a variant thereof in which such activity or activities are inhibited).
  • the method provided herein inhibits (e.g., by at least or more than 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%) or prevents displacement of the second nucleotide probe from the target RNA.
  • the displacement of the second polynucleotide probe can be measured using any method known in the art or described herein.
  • the first polynucleotide and/or the second polynucleotide probe comprises 1, 2, 3 or more non-natural nucleotides. In some embodiments, the first polynucleotide and/or the second polynucleotide probe comprises 4, 5, 6 or more non-natural nucleotides. In some embodiments, the non-natural nucleotides are selected from one or more of: locked nucleic acid (LNA), Super T (5-hydroxybutynl-2’-deoxyuridine), and Super G (8-aza- 7-deazaguanosine). In some embodiments, the first polynucleotide and/or the second polynucleotide probe comprises 1, 2, 3 or more locked nucleic acids (LNAs).
  • LNA locked nucleic acid
  • LNA locked nucleic acid
  • Super T 5-hydroxybutynl-2’-deoxyuridine
  • Super G 8-aza- 7-deazaguanosine
  • the second polynucleotide probe comprises at least one locked nucleic acid. In some embodiments, the first polynucleotide probe comprises at least one locked nucleic acid. In some embodiments, at least the first three positions of the second polynucleotide probe are LNA nucleotides. In some embodiments, at least the first three positions of the first polynucleotide probe are LNA nucleotides.
  • step b) and step c) of the method provided herein occur simultaneously by contacting the sample with the engineered reverse transcriptase disclosed herein and a ligase at the same time. In some embodiments, step b) and step c) of the method provided herein occur sequentially.
  • the method comprises, after step c), a step of binding the first polynucleotide probe and the second polynucleotide probe to a barcoded oligonucleotide.
  • the barcoded oligonucleotide is a spatially barcoded oligonucleotide.
  • the method comprises after step c), amplifying the cDNA.
  • the amplifying is performed by real-time quantitative PCR.
  • the method further comprises sequencing the cDNA.
  • the sequencing is next generation sequencing (NGS).
  • the method comprises permeabilizing the sample before contacting the sample with the first and/or second polynucleotide probes.
  • the sample used in the method described herein is a fixed sample.
  • the fixed sample is formalin-fixed.
  • the fixed sample is formalin-fixed and paraffin-embedded (FFPE).
  • the method described herein does not comprise using a third polynucleotide probe to gap-fill the sequence between the first polynucleotide probe and the second polynucleotide probe.
  • the sample used in the method described herein is a single cell, and/or the target RNA is the target RNA of a single cell.
  • the sample is a tissue.
  • the target RNA is the transcriptome of a sample.
  • the method described herein is for sequencing and/or genotyping a transcriptome comprising the target RNA. In some embodiments, the method provides spatial location of the target RNA. In some embodiments, the method described herein is for assessing, detecting the presence of or identifying a mutant allele, a single nucleotide variation, a splice isoform, or a TCR/BCR junction in the target RNA. In some embodiments, the method is for providing spatial location and/or single cell quantification of the mutant allele, the single nucleotide variation, the splice isoform or the TCR/BCR junction.
  • the method described herein is for single cell transcriptome analysis, optionally wherein the sample is a single cell, and the target RNA is transcriptome of the single cell.
  • the single cell transcriptome analysis is single cell CRISPR screening. In some embodiments, the single cell transcriptome analysis is single cell T-cell receptor (TCR) and/or B-cell receptor (BCR) sequencing.
  • TCR T-cell receptor
  • BCR B-cell receptor
  • the sample used in the method described herein is a cell or population of cells from a tissue or organ associated with or affected by a disease or disorder in a subject.
  • FIG. 1A is a schematic showing the domains of an engineered reverse transcriptase having two point mutations Y64A and D524A.
  • FIG. IB is a series of schematic diagrams showing methods described herein for profiling a target RNA sequence, including identifying mutations present therein.
  • FIG. 1C is a schematic showing a wild-type reverse transcriptase displacing a polynucleotide probe, and an engineered reverse transcriptase with no strand displacement activity leaving the second polynucleotide probe annealed to the target RNA.
  • FIG. 2 is a schematic showing the extension of a cDNA with a wild-type reverse transcriptase having strand displacement activity compared to extension of cDNA using an engineered reverse transcriptase with no strand displacement activity.
  • FIG. 3 is a schematic showing exemplary positions of a pair of primers, External FW and Probe RV.
  • FIG. 4A is an image of a protein gel showing production of the Stradivari-RT enzyme.
  • FIG. 4B is a graph showing relative fold change of cDNA produced using the engineered reverse transcriptase as described in Fig. 4A compared to using wild-type reverse transcriptase (SuperScriptll or SSII). Compared to commercially available enzymes, Stradivari- RT had reduced performance.
  • FIG. 5 is a graph showing relative fold change of cDNA produced using the engineered reverse transcriptase as described in Fig. 4A where LNA nucleotides were introduced in the RHS probe, as described in Example 1. The addition of a block reduced Stradivari activity.
  • FIG. 6 is a schematic showing the extension of a cDNA with a wild-type reverse transcriptase having strand displacement activity compared to extension of cDNA using an engineered reverse transcriptase with a substantially disrupted or abolished strand displacement activity.
  • the design of RHS and LHS probes is shown.
  • Exemplary positions of a pair of primers, External FW and Probe RV, used to amplify a cDNA extended using a wild-type reverse transcriptase are also shown.
  • FIG. 7 is a graph showing relative fold change of cDNA produced using the RHS and LHS probes, reverse transcriptases (SuperScriptll or SSII) and primers as described in Example 1.
  • FIG. 8 is a schematic showing the extension of a cDNA with an engineered reverse transcriptase and ligation of the cDNA to the second polynucleotide probe using SplintR ligase. Primers used for amplifying produced cDNA in both reaction conditions are shown. The schematic shows testing if gap-filling and ligation can be used in a single step.
  • FIG. 9 is a pair of graphs showing fold change production of cDNA using a reverse transcriptase with strand displacement activity, an engineered reverse transcriptase without strand displacement activity, and simultaneous reverse transcription and ligation.
  • qPCR- amplified cDNA product is graphed for each condition. The data demonstrate that gap-filling and ligation could be incorporated in one single step, that SDV-RT elongation is blocked by the presence of the RHS probe and that the two probes can be ligated after gap filling with high efficiency.
  • FIG. 10A is a Uniform Manifold Approximation and Projection (UMAP) plot showing clustering of single-cell RNA-sequencing data of two cell lines, HEL (erythroblast cell line) and CCRF (T lymphoblast cell line), in which two highly expressed genes GAPDH and CTCF were detected as described in Example 2.
  • the RNA projected UMAPs successfully separated the cell lines, demonstrating that Stradivari-RT does not interfere with the standard lOx kit for singlecell RNA-seq.
  • FIG. 10B is a pair of graphs showing the number of RNA features and RNA counts measured using single-cell RNA-sequencing of two cell lines, HEL and CCRF, in which two highly expressed genes GAPDH and CTCF were detected as described in Example 2.
  • FIG. 10C is a graph showing the expression of specific cell-line marker genes, identifying the pattern of expression of lymphoid genes in CCRF cells and erythroid genes in HEL cells as described in Example 2.
  • FIG. 10D is a UMAP plot showing detected cDNA in single cells using polynucleotide probes with complementarity against target GAPDH transcripts, with a 9-nucleotide third sequence space between the first polynucleotide probe and the second polynucleotide probe. Signal was successfully detected in single cell.
  • FIG. 10E is a UMAP plot showing detected cDNA in single cells using polynucleotide probes with complementarity against target CTCF transcripts, with a 9-nucleotide third sequence space between the first polynucleotide probe and the second polynucleotide probe. Signal was successfully detected in single cell with 100% genotyping accuracy as shown by the LOGO sequence plots.
  • FIG. 10F is a schematic showing the sequences of the two 9-nucleotide GAPDH and CTCF sequences identified using an engineered reverse transcriptase.
  • FIG. 11 is a series of UMAP plots showing the expression levels of specific cell-line marker genes (GATA1, GYPA, CD3D, and CD3G), identifying the pattern of expression of lymphoid genes in CCRF cells and erythroid genes in HEL cells as described in Example 2.
  • GATA1, GYPA, CD3D, and CD3G specific cell-line marker genes
  • FIG. 12A is a UMAP plot showing clustering of single-cell RNA-sequencing data of two cell lines, HEP2G (a BRAF WT cell line) and SKML (a BRAFV600E heterozygous mutant cell line) as described in Example 3. It was found that Stradivari can be used for accurate genotyping of cell lines. Prior technologies failed to profile this mutant locus. The RNA projected UMAPs successfully separated the cell lines, demonstrating that Stradivari-RT does not interfere with the standard lOx kit for single-cell RNA-seq.
  • FIG. 12B is a series of graphs showing the number of RNA features an RNA counts measured using single-cell RNA-sequencing of two cell lines, HEP2G (WT) and SKML (Het), as described in Example 3.
  • the number of RNA features and RNA counts were concordant with lOx genomics guidelines, underscoring the compatibility of Stradivari-RT with lOx application.
  • FIG. 12C is a heat map graph showing the pattern of expression of hepatic and melanoma genes measured using single-cell RNA sequencing of two cell lines, HEP2G (WT) and SKML (Het), as described in Example 3. The majority of HEP2G cells were genotyped as BRAF wild-type (>90%).
  • FIG. 12D is a UMAP plot showing genotyping of HEP2G cells and SKML cells, as described in Example 3. With regards to the SKML line, heterozygous, mutant and wild type cells were detected, likely the result of allelic drop out.
  • FIG. 13A is a series of UMAP plots showing detected cDNA in single cells using polynucleotide probes with complementarity against target CD5, MME, FCER2, MS4A1, CD22, ITGAX, IL2RA, and IL3RA transcripts from hairy cell leukemia (BRAF V600E mutant) cells, as described in Example 4.
  • FIG. 13B is a UMAP plot showing clustering of single-cell RNA-sequencing data of cells from a patient sample with Hairy cell leukemia cells, in which the BRAFV600E mutation was detected as described in Example 4. The vast majority of BRAFV600E mutant cells fell within the hairy cell leukemia cluster.
  • FIG. 13C is a pair of UMAP plots showing two distinct clusters (a high CD38 expression/low FMOD expression cluster and a low CD38/high FMOD expression cluster) identified using single-cell RNA-sequencing data measured in a patient sample chronic lymphocytic leukemia (CLL) as described in Example 4.
  • CLL chronic lymphocytic leukemia
  • FIG. 13D is a UMAP plot showing that in the two clusters, the high CD38 expression/low FMOD expression cluster and the low CD38/high FMOD expression cluster, the majority of BTK mutant cells were found within the low CD38/high FMOD expression cluster as described in Example 4.
  • FIG. 14A is a histological image of a bone marrow tissue sample from a patient with NPM1 mutant acute myeloid leukemia (AML). The histological image was stained with an antibody against the mutant NPM1 protein as described in Example 5.
  • FIG. 14B is a probe-based spatial transcriptomic image of a bone marrow tissue sample from a patient with NPM1 mutant acute myeloid leukemia (AML) obtained as described in Example 5.
  • the NPM1 mutational landscape as defined by sequencing was compared to the mutational landscape defined by an antibody against the mutant NPM1 protein.
  • the term “about” refers to a range of +/- 10% of the stated value. In some embodiments, the range is +/- 5%, +/- 3%, +/- 2%, +/- 1%, +/- 0.5%, or +/- 0.1% of the stated value.
  • locked nucleic acid or “LNA” refers to a modified RNA nucleotide in which the ribose moiety is modified with a bridge connecting the 2’ oxygen and the 4’ carbon.
  • Described herein is a new method for genotyping the transcriptome with single-cell and spatial resolution using commercially available probe-based techniques.
  • the new method utilizes a new engineered reverse transcriptase, and it unexpectedly allows simultaneous identification of single nucleotide variations in the RNA as well as profiling the entire transcriptome in fixed samples.
  • the inventors designed and engineered a Reverse-Transcriptase enzyme that can copy RNA sequences from a template strand into a probe set.
  • probe-based single-cell and spatial products such as lOx Genomics FFPE Visium or lOx Genomics single-cell Flex kits.
  • Probe-based single-cell and spatial products such as lOx Genomics FFPE Visium or lOx Genomics single-cell Flex kits, are well known in the art.
  • the Single Cell Gene Expression Flex Fixed RNA Profiling assay and Spatial Gene Expression for FFPE use probes that target protein-coding genes in the human or mouse transcriptome. Each probe consists of a pair of oligonucleotides that hybridize to the targeted transcript and are subsequently ligated. Briefly, human or mouse whole transcriptome probe panels, consisting of a pair of specific probes for each targeted gene, are added to the tissue or permeabilized fixed single cells. These contiguous probe pairs hybridize to their gene target and are then ligated to one another.
  • the ligated probe pairs bind with spatially barcoded oligonucleotides present on the Capture Area for spatial analysis, or are encapsulated in oil droplets and barcoded for single-cell analysis. All the probes captured by primers on a specific spot or droplet share a common barcode. Libraries are generated from the probes and sequenced and the spatial/cell barcodes are used to associate the reads back to the tissue section images for spatial mapping of gene expression or to a single cell. This powerful tool allows for gene expression profiling in fixed cells and tissues. Since the probe set is sequenced instead of the RNA, this approach can profile the transcriptome in samples where the RNA is highly degraded, such as fixed samples.
  • any possible information about the original RNA sequence including point mutations, small deletions, or duplications is lost. Accurately retrieving this information is essential for evaluating how these variants (for example, cancer driver mutations) affect the cellular phenotype.
  • the invention described herein aims to overcome this limitation.
  • the inventors developed a methodology that, using a gap-filling approach, allows to retain all the benefits of this assay, while adding additional layers of information about the original RNA nucleotide composition.
  • the inventors used non-contiguous locusspecific probes, separated from each other by a small gap, e.g., from 6 to 20 base pairs.
  • the inventors used a reverse-transcriptase enzyme to fill this gap, copying the original RNA sequence that is located between the two probes. After ligation, the two probes together with the newly copied sequence form a single DNA molecule, which can be captured and barcoded by single-cell or spatial assays. In this way, when a mutation is present on the original RNA molecule, it will be incorporated in the probe set and sequenced by NGS, without the need to design mutation- specific probes.
  • RT enzymes possess very high strand displacement activity, which is a crucial feature in their function during reverse transcription. This feature represents a major limitation for workflow of this methodology, as the RT enzyme synthesizes the DNA strand and fills the gap in between probes. When the enzyme reaches the second probe, it can displace it, continuing to copy the RNA molecule without stopping, resulting in the misincorporation of the copied sequence into the capturable molecule (see attached overview) and loss of the barcode on one of the probes.
  • M-MLV RT Moloney Murine Leukemia Virus Reverse Transcriptase
  • This enzyme allows to incorporate a copy of the original RNA sequence into a locus-specific probe set, enabling genotyping of the transcriptome in fixed samples, providing spatial location and single-cell quantification of the mutant allele to study phenotypic changes of known driver mutations with high resolution.
  • the reverse transcriptase engineered as described herein is derived from any source, e.g., Moloney Murine Leukemia Virus.
  • Various reverse transcriptases that can be engineered as described herein (to interfere with or abolish the strand displacement activity and/or RNAse-H activity), and used in the methods described herein, are known in the art.
  • the reverse transcriptase is any natural reverse transcriptase.
  • the reverse transcriptase is HIV-1 reverse transcriptase, M-MLV reverse transcriptase, AMV reverse transcriptase, or Telomerase reverse transcriptase.
  • an engineered reverse transcriptase in which the strand displacement activity is inhibited, substantially abolished or entirely abolished, e.g., by introducing a mutation (e.g., amino acid substitution) in the first finger domain of the reverse transcriptase.
  • a mutation e.g., amino acid substitution
  • a mutation is a substitution or substitution of one or more amino acids (in the finger domain and/or the RNAse-H domain of a reverse transcriptase). In some embodiments, a mutation comprises a deletion or insertion of one or more amino acids (in the finger domain and/or the RNAse-H domain of a reverse transcriptase). In some embodiments, multiple mutations can be introduced (in the finger domain and/or the RNAse-H domain of a reverse transcriptase), e.g., for optimal strand displacement activity and/or interference with RNAse-H activity. In some embodiments, a mutation is a deletion or insertion (in the finger domain and/or the RNAse-H domain of a reverse transcriptase).
  • a mutation is a partial truncation of the first finger domain and/or the RNAse-H domain of a reverse transcriptase.
  • a mutation of a reverse transcriptase is a mutation in Y64, e.g., Y64A or another mutation in Y64 that can interfere with the strand displacement activity.
  • a mutation of a reverse transcriptase is a mutation in D524, e.g., D524A, D524G, D524N or another mutation in D524 that can interfere with the RNAse-H activity.
  • a mutation of a reverse transcriptase is a mutation in D583, e.g., D583N or another mutation in D583 that can interfere with the RNAse-H activity.
  • a mutation of a reverse transcriptase is a mutation in E562, e.g., E562Q or another mutation in E562 that can interfere with the RNAse-H activity.
  • the engineered reverse transcriptase comprises more than one (e.g., 2 or 3) mutations that interfere with the RNAse-H activity (such as 2 or 3 mutations described herein).
  • an engineered reverse transcriptase comprising Y64 mutation in Moloney Murine Leukemia Virus reverse transcriptase, or comprising equivalent mutations(s) in a reverse transcriptase from different species.
  • an engineered reverse transcriptase comprising D524, E562, D583 or partial truncation of the RNAse-H domain in Moloney Murine Leukemia Virus reverse transcriptase, or comprising equivalent mutations(s) in a reverse transcriptase from different species.
  • an engineered reverse transcriptase comprising Y64A mutation and/or D524A mutation in Moloney Murine Leukemia Virus reverse transcriptase, or comprising equivalent mutations(s) in a reverse transcriptase from different species.
  • an engineered reverse transcriptase comprising D524A, D524G, D524N, E562Q, D583N or partial truncation of the RNAse-H domain in Moloney Murine Leukemia Virus reverse transcriptase, or comprising equivalent mutations(s) in a reverse transcriptase from different species.
  • the engineered reverse transcriptase described herein disrupts the ability to displace the second polynucleotide probe by at least 70%, 75%, 80%, 85%, 90%, 98%, 99% or 100%.
  • the mutation(s) described herein are in the sequence of M-MLV reverse transcriptase of SEQ ID NO: 1.
  • the engineered reverse transcriptase comprises, essentially consists of, or consists of SEQ ID NO: 2 or a variant thereof (e.g., a variant having one, two or more mutations described herein).
  • the engineered reverse transcriptase comprises, essentially consists of, or consists of SEQ ID NO: 2 or a variant thereof (e.g., a variant having one, two or more mutations described herein), and further comprises one or more amino acids before SEQ ID NO: 2 that, e.g., may facilitate protein production (e.g., an amino acid methionine or M).
  • compositions comprising an engineered reverse transcriptase described herein.
  • nucleic acids encoding the engineered reverse transcriptase described herein.
  • the ligase is any ligase known in the art that is capable of ligating DNA or cDNA.
  • the ligase is a DNA ligase capable of ligating singlestranded DNA molecules (e.g., wherein a single-stranded DNA molecule is annealed to an RNA molecule).
  • the ligase is a SplintR Ligase, also known as PBCV- 1 DNA Ligase or Chlorella virus DNA Ligase.
  • the ligase is a 10X Genomics kit ligase.
  • the first and second sequence are separated by 1 to 100 nucleotides. In some embodiments, the first and second sequence are separated by 3 to 40 nucleotides, or 6 to 20 nucleotides. In some embodiments, the first and second sequence are separated by 6 to 75 nucleotides, or 8 to 50 nucleotides. In some embodiments, the first and second sequence are separated by 8 to 40 nucleotides, or 9 to 20 nucleotides (e.g., 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides). In some embodiments, these nucleotides separating first and second sequences represent a gap between the right-hand side (RHS) and left-hand side (LHS) probe used in the methods described herein.
  • RHS right-hand side
  • LHS left-hand side
  • polynucleotide probe design methodology is also described herein (see, e.g., the examples and figures).
  • the polynucleotide probes are designed in accordance with 10X Genomics guidelines or design criteria for Visium spatial gene expression and/or chromium single cell gene expression flex.
  • the first polynucleotide probe comprises a nucleotide sequence substantially complementary to a first sequence of the target RNA and a second polynucleotide probe comprises a nucleotide sequence substantially complementary to a second sequence of the target RNA, wherein the nucleotide sequence of one or each probe that is substantially complementary to the target RNA is about 15 to about 30 nucleotides in length, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or any number of nucleotides in between any of these values.
  • “substantially complementary” is at least or more than 80%, 85%, 90%, 95% or 100% complementary.
  • “substantially complementary” allows for up to 1, 2, 3 or 4 mismatches (e.g., up to 2 mismatches). In some embodiments, “substantially complementary” is entirely complementary such that there are no mismatches between the part of the polynucleotide sequence that recognizes the target RNA and the target RNA.
  • the first polynucleotide and/or the second polynucleotide probe comprises one or more (e.g., 2, 3, 4 or 5 or more) non-natural nucleotides, e.g., locked nucleic acid (LNA), Super T (5-hydroxybutynl-2’-deoxyuridine), and/or Super G (8-aza-7- deazaguanosine).
  • LNA locked nucleic acid
  • Super T 5-hydroxybutynl-2’-deoxyuridine
  • Super G 8-aza-7- deazaguanosine
  • the first polynucleotide and/or the second polynucleotide probe comprises 1, 2, 3, 4 or 5 or more locked nucleic acids (LNAs), optionally wherein the second polynucleotide probe comprises 1, 2, 3, 4, 5 or more LNAs.
  • LNAs locked nucleic acids
  • the first polynucleotide probe is referenced as the LHS probe.
  • the second polynucleotide probe is referenced as the RHS probe.
  • locked nucleic acid (LNA) nucleotides are to be used in the first 1, 2, 3 or 4 positions (e.g., in the first three positions) of the RHS probe.
  • the 3’ most nucleotide of the gap between the probes is a T. In some embodiments, the 3’ most nucleotide of the gap between the first sequence and the second sequence of the target RNA is a T.
  • the RHS probe is 5’ phosphorylated.
  • the GC content of one or each of the polynucleotide probes to be used in the methods described herein is from 44% to 72%.
  • the GC content of the RHS polynucleotide probe is at least 44%, less than 72%, or from 44% to 72%.
  • the GC content of the LHS polynucleotide probe is at least 44%, less than 72%, or from 44% to 72%.
  • the GC content of the first polynucleotide probe is at least 44%, less than 72%, or from 44% to 72%.
  • the GC content of the second polynucleotide probe is at least 44%, less than 72%, or from 44% to 72%.
  • the sample is permeabilized before contacting the sample with the first and/or second polynucleotide probes.
  • the sample is a fixed sample (e.g., formalin-fixed and paraffin-embedded (FFPE)).
  • the method described herein does not comprise using a third polynucleotide probe to gap-fill the sequence between first polynucleotide probe and the second polynucleotide probe.
  • the methods described herein use a reverse transcriptase (such as the engineered reverse transcriptase described herein) to serve the gap-filling function.
  • the first polynucleotide probe and the second polynucleotide probe can be any probes described herein or known in the art for use for similar purposes (such as single-cell and spatial transcriptomics).
  • the probes can be barcoded or can be bound to a barcoded oligonucleotide (e.g., spatially barcoded).
  • the probes that can be used for single-cell and/or spatial transcriptomics are those made in accordance with the methods known in the art by, e.g., 10X Genomics.
  • the methods described herein are used for genotyping a transcriptome comprising the target RNA.
  • the methods described herein are used for sequencing a transcriptome comprising the target RNA.
  • the methods describing herein is for genotyping and/or sequencing the entire transcriptome of a single cell.
  • the methods described herein are used for single cell transcriptome analysis such as single cell CRISPR screening, single cell T-cell receptor (TCR) sequencing, and/or single cel B-cell receptor (BCR) sequencing.
  • the methods described herein are for identifying a mutation, a single nucleotide variation, a splice isoform, and/or a TCR/BCR junction in the target RNA (e.g., on a single cell level).
  • the methods described herein are for identifying a mutation such as a single nucleotide variation in the target RNA (e.g., on a single cell level). In some embodiments, the methods described herein provide spatial location of the target RNA and/or mutation, single nucleotide variation, splice isoform or TCR/BCR junction (e.g. on a single cell level).
  • Example 1 An engineered reverse transcriptase described herein (Stradivari-RT) enables gap-filling reactions of non-contiguous probes
  • FFPE formalin-fixed and paraffin-embedded
  • probe-based droplet and spatial workflows were adapted to selectively profile point mutations, deletions, or duplications in archival tissues for combined genotyping and phenotypic profiling.
  • the inventors hypothesized that by gap-filling between two non-contiguous probes targeting RNA molecules around a specific mutation, they could copy the original RNA sequence into the probe set for downstream genotyping.
  • M-MuLV reverse transcriptase was engineered, knocking out its strand displacement function to enable a gap-filling reaction without loss of probe binding (FIG. 2).
  • Stradivari-RT engineered Strand displacement variant Reverse Transcriptase
  • Genotyping of Transcriptomes by Stradivari turns the admixture of mutant and wild-type cells from a limitation to an advantage, enabling the direct comparison of the molecular or spatial features of mutant versus wild-type cells in archival samples, within the same individual, overcoming patient-specific confounders in human studies.
  • FFPE paraffin-embedded
  • FFPE specimens present challenges when it comes to analyzing RNA.
  • the RNA within these samples is highly fragmented, with susceptibility to further degradation under suboptimal storage conditions.
  • the loss of poly- A tails adds another layer of complexity, which limits the usefulness of oligo-dT primed reverse transcription.
  • imaging-based platforms like MERSCOPE (Vizgen), CosMx (Nanostring), and Xenium (lOx Genomics) have demonstrated spatial mapping of hundreds or thousands of genes in unfixed samples, they are constrained by their reliance on a targeted panel for gene expression analysis with limited discovery power to profile diverse RNA species or unknown sequences.
  • the Visium (lOx Genomics) chemistry for FFPE samples also relies on a predefined panel to target and capture RNA fragments, enabling near transcriptome-level measurement of gene expression, yet still confined to quantifying the expression of known protein-coding genes rather than base-by-base sequencing of RNA.
  • the Single Cell Gene Expression Flex Fixed RNA Profiling assay and Spatial Gene Expression for FFPE use probes that target protein-coding genes in the human or mouse transcriptome. Briefly, human or mouse whole transcriptome probe panels, consisting of a pair of specific probes for each targeted gene, are added to the tissue or permeabilized fixed single cells. These contiguous probe pairs hybridize to their gene target and are then ligated to one another. Subsequently, the ligated probe pairs bind with spatially barcoded oligonucleotides present on the Capture Area for spatial analysis, or are encapsulated in oil droplets and barcoded for single-cell analysis. All the probes captured by primers on a specific spot or droplet share a common barcode.
  • the approach described herein uses non-contiguous locus-specific probes, separated from each other by a small gap, from 6 to 20 base pairs.
  • a reversetranscriptase enzyme is used to fill this gap, copying the original RNA sequence that is located between the two probes.
  • the two probes together with the newly copied sequence form a single DNA molecule, which can be captured and barcoded by single-cell or spatial assays.
  • NGS next generation sequencing
  • RT reverse transcriptase
  • M-MuLV RT Moloney Murine Leukemia Virus Reverse Transcriptase
  • the Strand displacement variant Reverse Transcriptase (Stradivari-RT) engineered as described herein allowed to incorporate a copy of the original RNA sequence into a locusspecific probe set, enabling genotyping of the transcriptome in fixed samples, providing spatial location and single-cell quantification of the mutant allele to study phenotypic changes of known driver mutations with high resolution.
  • a mutant version of M-MLV reverse transcriptase was engineered and produced by introducing two point mutations to knock out 2 specific functions of the enzyme.
  • the Y64A mutation was introduced in the first finger domain of the protein to delete strand displacement activity, while D524A was introduced in the RNAse-H domain of the enzyme to avoid RNA degradation during reverse transcription (FIG. 1A).
  • Stradivari-RT was tested in a targeted cDNA experiment.
  • Total RNA was obtained from K562 cells and GAPDH-specific reverse transcription was performed with increasing concentrations of Stradivari-RT or SuperScriptll (wild-type control) (FIG. 1C, FIG. 4A and FIG. 4B).
  • the efficiency of the reaction was measured by real-time quantitative PCR with a set of primers designed on the GAPDH transcript (FIG. 3). It was observed that Stradivari-RT has slightly lower performance compared to the commercially available enzyme; at the highest concentration tested, a 50% reduction in GAPDH cDNA level was observed for Stradivari-RT most likely due to its inability to resolve the secondary structure of the RNA (FIG.
  • the LHS probe serves as a primer to initiate the reverse transcription, and after gap filling the reverse transcriptase can either displace the RHS probe, elongating the cDNA molecule passing through it, or, with effective knockout of the strand displacement function, stop as soon it reaches the 5’ phosphate nucleotide of the RHS probe and eventually lose the binding with the newly synthesized cDNA (FIG. 1C).
  • cDNA synthesis was performed with a LHS-like probe targeting the NPM1 transcript, in the presence or absence of the blocking RHS probe. The reaction was performed with Stradivari-RT or SuperScript II, as a negative control.
  • FIG. 8 Two non-contiguous probes targeting GAPDH mRNA, separated by 9 nucleotides, were pre-annealed to total RNA obtained from K562 cells. After annealing, Stradivari-RT or Superscript! plus SplintR ligase were added to initiate reverse transcription and simultaneously ligate the two probes after the gap-filling reaction. Reverse transcription blocking efficiency was tested as described above, while ligation efficiency was tested by qPCR using primers targeting both probe handles. Inventors confirmed that Stradivari- RT elongation is blocked by the presence of the second probe (FIG. 9) and, more importantly, that the two probes can be ligated after gap filling with high efficiency (FIG. 9).
  • Example 2 Single-cell probes gap-filling technology using an engineered reverse transcriptase (Stradivari-RT)
  • a cell line mixing experiment was performed with HEL (erythroblast cell line) and CCRF (T lymphoblast cell line) cells. Cells were fixed and permeabilized, and permeabilized cells were incubated for 24 hours with lOx Genomics human whole-transcriptome probe set supplemented with custom designed probe pairs targeting two highly expressed genes, GAPDH and CTCF. Unlike lOx Genomics gene-specific probe pairs that are designed to be contiguous on the matched mRNA, for this experiment custom probe pairs were designed with a 9- nucleotide gap between the LHS and RHS.
  • RNA projected UMAPs successfully separated the cell lines, demonstrating that Stradivari-RT does not interfere with the standard lOx kit for single-cell RNA-seq (FIG. 10A).
  • the number of RNA features and RNA counts were concordant with lOx genomics guidelines, underscoring the compatibility of Stradivari-RT with lOx application (FIG. 10B).
  • the expression of specific cell-line marker genes was examined, identifying the expected pattern of high expression of lymphoid genes in CCRF cells and erythroid genes in HEL cells (FIG. IOC and FIG. 11).
  • the DNA sequence coding for M-MLV reverse transcriptase harboring Y64A and D524A mutation was synthesized as a gene fragment (IDT) flanked by restriction enzyme sites Xbal and Spel.
  • pTXBl plasmid and gene fragments were digested with Xbal (NEB, R0145S) and Spel (NEB, R3133S) Ih at 37°C.
  • the digested plasmid backbone was purified with gel extraction (QIAquick Gel Extraction Kit, #28704), and dephosphorylated with Shrimp Alkaline Phosphatase (NEB, M0371S) at 37°C for 30 min.
  • the digested gene fragments were purified with columns (QIAquick PCR Purification Kit, #28106).
  • the purified gene fragments and plasmid backbones were ligated with quick ligase (NEB, M2200) at room temperature for 30 min and subsequently transformed into competent cells per vendor instruction (NEB 5-alpha, NEB C2987H).
  • StraDiVari-RT production
  • IPTG isopropyl-P-D-1- thiogalactopyranoside
  • Protein was dialyzed in five dialysis steps using 15 mL of 2x dialysis buffer (100 HEPES-KOH pH 7.2, 0.2 M NaCl, 0.2 mM EDTA, 2 mM DTT, 20% glycerol) and concentrated to 1 mL by centrifugation at 5,000g. The protein concentrate was transferred to a new tube and mixed with an equal volume of 100% glycerol. nb-Tn5 aliquots were stored at -80°C.
  • 2x dialysis buffer 100 HEPES-KOH pH 7.2, 0.2 M NaCl, 0.2 mM EDTA, 2 mM DTT, 20% glycerol
  • K562 cells were acquired from the American Type Culture Collection (CCL-243). OCI- AML3 cells were donated. OCI-AML3 cells were maintained at 37 °C and 5% CO2 in alpha- MEM medium (catalog number) supplemented with 10% FBS (Thermo Fisher Scientific, 16000044). K562 cells were maintained at 37C and 5% CO2 in R10 medium (RPMI with stabilized L-glutamine (Thermo Fisher Scientific, 11875119) supplemented with 10% FBS.
  • Example 3 Genotyping single cells using an engineered reverse transcriptase (Stradivari- RT) This example shows that Stradivari-RT can accurately genotype single cells.
  • Two cell lines were used: 1) HEP2G, a BRAF WT cell line and 2) SKML, a BRAFV600E heterozygous mutant cell line. Prior technologies developed in the lab failed to profile this mutant locus.
  • the RNA projected UMAPs successfully separated the cell lines, demonstrating that Stradivari-RT does not interfere with the standard lOx kit for single-cell RNA-seq (FIG. 12A).
  • RNA features and RNA counts were concordant with lOx genomics guidelines, underscoring the compatibility of Stradivari-RT with lOx application (FIG. 12B).
  • the expression of specific cell-line marker genes was examined, identifying a pattern of expression of hepatic and melanoma genes (FIG. 12C).
  • the majority of HEP2G cells were genotyped as BRAF wild-type (>90%) (FIG. 12D and Table 1).
  • SKML line heterozygous, mutant, and wild type cells were detected, likely the result of allelic drop out (FIG. 12D and Table 1).
  • Custom targeted probes were designed so that there was a 9-20 base gap between the right-hand side (RHS) and left-hand side (LHS) probe. Per lOx recommendations for custom probe design, a GC content between 44 - 72% for each probe half was achieved. Gap size and location was adjusted so that when possible the 3' most nucleotide of the gap would be a T.
  • the handle for the RHS probe was constructed depending on whether the intended use was for 10X FLEX single cell or 10X Visium spatial protocol. For the LHS probe, the Nextera read 2 sequence was used for the handle.
  • Cells were processed according to the lOx protocol for the fixation of cells and nuclei. Briefly, suspensions of up to 10 million cells were spun-down at 400 ref for 5 min at 4C and cells were resuspended in 1 mL of Fixation Buffer (4% Formaldehyde, IX Cone. Fix and Perm Buffer - lOx Genomics PN-2000517). Cells were fixed for 30 min at room temperature. To stop the fixation, cells were spun-down at 850 ref for 5 min at room temperature and quenched with 1 mL of Quenching Buffer (IX Cone. Quench Buffer - lOx Genomics PN-2000516).
  • Fixation Buffer 4% Formaldehyde, IX Cone. Fix and Perm Buffer - lOx Genomics PN-20005157
  • Hybridizations were set up in 80 pl of hybridization mix with 20 pl of Human WTA probes (lOx Genomics PN-2000510 or PN- 2000718). To use custom probes, a spike-in pool containing 40 nM of each LHS and 80 nM of each RHS probe in nuclease-free water was prepared. 5 pl of custom probe mix was added to the hybridization mix. Hybridizations were performed at 42C for 16-24h. After hybridization samples were washed 3 times in Post-Hyb Wash Buffer for 10 min at 42C. After the washes cells were resuspended in Post-Hyb Resuspension Buffer, filtered through a Miltenyi Biotec 30 um filter and measured with the cell counter to determine the amount needed for the Chromium X run.
  • lOx Genomics protocol and guidelines were followed regarding the volume of cells and reagents required per well according to the targeted cell recovery.
  • lOpl of StraDiVari enzyme was added and the volume of the Post-Hyb Resuspension Buffer was reduced by the same amount in the cell-reagent mix.
  • 20,000 cells per sample were targeted.
  • GEMs were recovered as indicated by lOx Genomics. Reverse transcription and ligation were performed at 25 °C for 90 minutes, followed by extension at 60°C for 45 minutes and enzyme deactivation at 80°C for 20 minutes.
  • the product After processing the GEMs, the product is pre-amplified; in addition to the Pre-Amp Primers B (lOx Genomics PN-2000529) 5pl of lOpM Nextera partial read 2 primer was added to amplify the targeted library. Sample indexing of gene expression library was performed per lOx protocol. For indexing of the targeted libraries, an additional 15 cycles of pre-amplification PCR was performed using a TruSeq partial R1 primer and a Nextera partial R2 primer followed by 5-10 cycles of indexing using a N7 NY sample indexing primer as well as an indexed P5-TruSeq R1 primer.
  • FIG. 13A-13B Hairy cell leukemia sample
  • CLL chronic lymphocytic leukemia
  • FIG. 13C-13D The cluster of hairy cell leukemia cells (FIG. 13 A) were able to be clearly identified.
  • CLL sample two distinct clusters of CLL cells were identified based on RNA expression.
  • Example 5 Determination of mutational landscape using an engineered reverse transcriptase (Stradivari-RT) and a spatial transcriptomic platform
  • FIG. 14A A bone marrow sample from a patient with NPM1 mutant acute myeloid leukemia (AML) was processed by combining Stradivari with lOx Visium spatial transcriptomic platform (FIGs. 14A and 14B).
  • the NPM1 mutational landscape as defined by sequencing (FIG. 14B) was compared to the mutational landscape defined by an antibody against the mutant NPM1 protein (FIG. 14A).
  • Targeted and gene expression cDNA libraries were prepared following the guidelines outlined in the Visium CytAssist Spatial Gene Expression for FFPE User Guide with the following modifications.
  • FFPE tissue sections of 5 pm thickness were mounted on Surgipath Apex Superior Adhesive Slides (LeicaTM). Sections underwent deparaffinization followed by H&E staining. Following imaging, sections were processed for hematoxylin de-staining and decrosslinking.
  • Probe hybridization was set up in 140.1 pL of FFPE Hyb Buffer (lOx Genomics PN 2000423), 20 pL of Human WT Probes v2 - RHS (lOx Genomics PN - 2000657), 20 pL of Human WT Probes v2 - LHS (lOx Genomics PN - 2000658), and 20 pL of custom probe.
  • FFPE Hyb Buffer LOx Genomics PN 2000423
  • 20 pL of Human WT Probes v2 - RHS lOx Genomics PN - 2000657
  • 20 pL of Human WT Probes v2 - LHS lOx Genomics PN - 2000658
  • custom probes a spike-in pool containing 25 nM of each LHS and 50 nM of each RHS probe in nuclease-free water was prepared. 20pL of custom probe mix was added to the hybridization mix. Hybridization was performed at 50°C

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Abstract

Selon certains aspects, la présente invention concerne des variants de transcriptase inverse ingénierisés permettant d'éviter le déplacement des brins et/ou la digestion des hybrides ADN:ARN, ainsi que des compositions et des utilisations associées. Selon certains aspects, la présente divulgation concerne des procédés d'analyse de transcrits d'ARN, par exemple dans des échantillons fixes, par l'utilisation, entre autres, de variants d'enzymes transcriptases inverses ingénierisés.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001068895A1 (fr) * 2000-03-15 2001-09-20 Invitrogen Corporation Transcriptases inverses haute fidelite et utilisations correspondantes
WO2020132966A1 (fr) * 2018-12-26 2020-07-02 深圳华大生命科学研究院 Transcriptase inverse ayant une activité enzymatique accrue et application de celle-ci
US20210332355A1 (en) * 2011-09-16 2021-10-28 Lexogen Gmbh Strand displacement stop (sds) ligation

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Publication number Priority date Publication date Assignee Title
WO2001068895A1 (fr) * 2000-03-15 2001-09-20 Invitrogen Corporation Transcriptases inverses haute fidelite et utilisations correspondantes
US20210332355A1 (en) * 2011-09-16 2021-10-28 Lexogen Gmbh Strand displacement stop (sds) ligation
WO2020132966A1 (fr) * 2018-12-26 2020-07-02 深圳华大生命科学研究院 Transcriptase inverse ayant une activité enzymatique accrue et application de celle-ci

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LOHMAN ET AL., NUCLEIC ACID RESEARCH, vol. 42, no. 3, 2013, pages 1831 - 1844
MASAKI MIZUNO ET AL: "Insight into the Mechanism of the Stabilization of Moloney Murine Leukaemia Virus Reverse Transcriptase by Eliminating RNase H Activity", BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY, vol. 74, no. 2, 1 January 2010 (2010-01-01), pages 440 - 442, XP055075589, ISSN: 0916-8451, DOI: 10.1271/bbb.90777 *
PAULSON ET AL: "Substitution of alanine for tyrosine-64 in the fingers subdomain of M-MuLV reverse transcriptase impairs strand displacement synthesis and blocks viral replication in vivo", VIROLOGY, ELSEVIER, AMSTERDAM, NL, vol. 366, no. 2, 14 September 2007 (2007-09-14), pages 361 - 376, XP022249497, ISSN: 0042-6822, DOI: 10.1016/J.VIROL.2007.04.028 *
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