Method for specific detection of nucleic acid sequences using in vitro transcription and in situ sequencing

Abstract

This disclosure generally relates to methods and compositions for detecting one or more target nucleic acid sequences (e.g., nucleic acid barcodes) in situ in biological samples. Fluorescence microscopy is one of the most important, widely used, and powerful imaging techniques in biomedical research. The spatial resolution of modern fluorescence microscopy has improved to the point where sub-diffraction-limit resolution is routinely possible. For various spatial biological applications, it is often desirable to visualize specific nucleic acid sequences in situ.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 508,353, filed on 15 June 2023, the disclosure of which is incorporated herein by reference for all purposes. [Background technology]

[0002] Fluorescence microscopy is one of the most important, widely used, and powerful imaging techniques in biomedical research. The spatial resolution of modern fluorescence microscopy has improved to the point where sub-diffraction-limit resolution is routinely achievable. For various spatial biological applications, visualizing specific nucleic acid sequences in situ is often desirable. However, current methods require a combination of numerous steps that result in low sensitivity for low-abundance nucleic acid species, particularly in cells with inherently low mRNA production. Such limitations are especially pronounced in methods such as fluorescence in situ sequencing (FISSEQ), where low sensitivity is a major obstacle. Therefore, it is desirable to develop new methods with higher sensitivity, especially for low-abundance nucleic acid species. [Overview of the project] [Means for solving the problem]

[0003] This disclosure generally relates to methods and compositions for in situ detection of one or more target nucleic acid sequences in a biological sample.

[0004] This specification discloses, in various embodiments, a method for determining in situ the presence, quantity, and / or localization of a nucleic acid sequence of interest in one or more fixed mammalian cells in a biological sample, the method comprising: (a) reacting in one or more fixed mammalian cells with (i) a DNA molecule containing the nucleic acid sequence of interest operably linked to a sequence-specific RNA polymerase promoter and (ii) a sequence-specific RNA polymerase to produce an RNA transcript of the nucleic acid sequence of interest; (b) reacting the RNA transcript in situ with reverse transcriptase to produce a cDNA molecule containing the nucleic acid sequence of interest; and (c) sequencing the cDNA molecule in situ to visualize the nucleic acid sequence of interest in one or more fixed mammalian cells. In some embodiments, the method comprises contacting the fixed mammalian cells with RNase to degrade endogenous RNA molecules prior to step (a).

[0005] In some embodiments, the DNA molecule is an exogenous nucleic acid molecule introduced into one or more mammalian cells before fixation, or is derived therefrom. In some embodiments, the nucleic acid sequence of interest is a barcoded polynucleotide. In some embodiments, the exogenous nucleic acid molecule is incorporated into the genome of one or more mammalian cells by viral transduction, site-specific nuclease, or site-specific recombinase. In some embodiments, the exogenous DNA molecule is introduced into one or more mammalian cells using a viral vector selected from lentiviral vectors, retroviral vectors, adenovirus vectors, HSV vectors, baculovirus vectors, virus-like particles, pseudotyped virus-like capsids, oncolytic virus vectors, or AAV vectors. In some embodiments, the exogenous nucleic acid sequence is incorporated at a specific site within the genome. In some embodiments, the exogenous nucleic acid sequence is incorporated at a random site within the genome. In some embodiments, the exogenous nucleic acid is not incorporated into the mammalian chromosome. In some embodiments, the exogenous nucleic acid molecule is retained in the nucleus of one or more mammalian cells. In some embodiments, the exogenous nucleic acid molecule is contained within a plasmid or artificial chromosome.

[0006] In some embodiments, the nucleic acid sequence of interest is an endogenous nucleic acid sequence, and the promoter is an exogenous promoter. In some embodiments, the endogenous nucleic acid sequence is variable between cells in a biological sample. In some embodiments, the endogenous nucleic acid sequence encodes a region containing a T cell receptor, a B cell receptor, an immunoglobulin sequence, a repetitive sequence, or a somatic mutation. In some embodiments, the nucleic acid sequence of interest is an endogenous sequence that does not change between cells in a biological sample.

[0007] In some embodiments, the DNA molecule is produced by reverse transcription using a DNA primer that hybridizes to a target RNA containing a nucleic acid sequence of interest in one or more fixed mammalian cells, the DNA primer comprising (i) a 5' nucleic acid sequence containing a sequence-specific RNA polymerase promoter, and (ii) a 3' nucleic acid sequence complementary to a portion of the target RNA adjacent to the nucleic acid sequence of interest. In some embodiments, the method further comprises converting the DNA molecule into double-stranded DNA by second-strand synthesis. In some embodiments, the 5' nucleic acid sequence of the DNA primer containing the sequence-specific RNA polymerase promoter is dsDNA. In some embodiments, the dsDNA is hybridized dsDNA or hairpin. In some embodiments, the target RNA molecule is digested whole or partially following the synthesis of the DNA molecule. In some embodiments, in-situ sequencing is sequencing by synthesis, sequencing by ligation, or sequencing by avidity. In some embodiments, in-situ sequencing is sequencing by synthesis.

[0008] In some embodiments, the sequence-specific RNA polymerase promoter is a phage promoter or a transcriptional variant thereof, and the sequence-specific RNA polymerase is a phage RNA polymerase. In some embodiments, the sequence-specific RNA polymerase promoter and sequence-specific RNA polymerase are selected from the group consisting of (i) a T7 promoter or a transcriptional variant thereof and a T7 RNA polymerase, respectively; (ii) a T3 promoter or a transcriptional variant thereof and a T3 RNA polymerase, respectively; and (iii) an SP6 promoter or a transcriptional variant thereof and an SP6 RNA polymerase, respectively. In some embodiments, the promoter is a T7 promoter and the RNA polymerase is a T7 RNA polymerase.

[0009] In some embodiments, the sequence-specific RNA polymerase promoter is a bacterial promoter, and the sequence-specific RNA polymerase is a bacterial RNA polymerase. In some embodiments, the sequence-specific RNA polymerase promoter is a eukaryotic promoter, and the sequence-specific RNA polymerase is a eukaryotic RNA polymerase. In some embodiments, the sequence-specific RNA polymerase promoter is a viral promoter, and the sequence-specific RNA polymerase is a viral RNA polymerase. In some embodiments, the sequence-specific RNA polymerase promoter is a synthetic promoter, and the sequence-specific RNA polymerase is a synthetic RNA polymerase.

[0010] In some embodiments, the DNA molecule further comprises a transcription terminator. In some embodiments, the transcription terminator is a T7 terminator. In some embodiments, the biological sample is fixed using a solution containing formaldehyde and / or paraformaldehyde. In some embodiments, the solution contains 4% paraformaldehyde. In some embodiments, the biological sample comprises a formalin-fixed paraffin-embedded (FFPE) sample containing one or more mammalian cells. In some embodiments, the biological sample is fixed by cryopreservation. In some embodiments, the sample comprises an optimal cleavage temperature compound, a hydrogel matrix, or a swellable polymer hydrogel. In some embodiments, the sample is fixed using a solution containing alcohol. In some embodiments, the alcohol is methanol or ethanol. In some embodiments, the sample is fixed using a solution containing glutaraldehyde.

[0011] In some embodiments, the nucleic acid sequence of interest is less than 100 nucleotides long, less than 90 nucleotides long, less than 80 nucleotides long, less than 70 nucleotides long, less than 60 nucleotides long, less than 50 nucleotides long, less than 40 nucleotides long, less than 30 nucleotides long, less than 25 nucleotides long, less than 20 nucleotides long, less than 15 nucleotides long, less than 10 nucleotides long, or less than 5 nucleotides long. In some embodiments, the DNA molecule further comprises one or more polynucleotide sequences encoding an exogenous protein, an endogenous protein, or a mixture of exogenous and endogenous proteins. In some embodiments, the DNA molecule further comprises one or more polynucleotide sequences encoding one or more exogenous proteins. In some embodiments, at least one subset of the one or more exogenous proteins is a synthetic protein and / or a chimeric protein. In some embodiments, the one or more exogenous proteins are independently selected from the group consisting of chimeric antigen receptors (CARs), antibodies, T cell receptors, cytokines, cell surface receptors, transcription factors, signaling proteins, and proteases. In some embodiments, two or more exogenous proteins are expressed. In some embodiments, the expression of an exogenous protein is controlled by an endogenous protein in one or more mammalian cells. In some embodiments, the DNA molecule further comprises a polynucleotide sequence encoding an endogenous protein. In some embodiments, the DNA molecule further comprises a polynucleotide sequence encoding an endogenous RNA. In some embodiments, the DNA molecule further comprises a polynucleotide sequence encoding an exogenous RNA. In some embodiments, the DNA molecule further comprises a polynucleotide sequence encoding a nucleic acid sequence that modifies the expression, function, and / or sequence of one or more genes. In some embodiments, the nucleic acid sequence that modifies the expression, function, and / or sequence of one or more genes is selected from the group consisting of sgRNA, gRNA, shRNA, and miRNA. In some embodiments, the DNA molecule further comprises a polynucleotide sequence encoding a viral genome. In some embodiments, the viral genome is an oncolytic viral genome.

[0012] In some embodiments, the DNA molecule includes a second sequence-specific RNA polymerase promoter configured to drive the transcription of a second nucleic acid sequence of interest in the presence of a second sequence-specific RNA polymerase. In some embodiments, the second nucleic acid sequence of interest is a second barcode polynucleotide. In some embodiments, the second sequence-specific RNA polymerase promoter and the second sequence-specific RNA polymerase are selected from the group consisting of (i) a T7 promoter or its transcriptional activity variant and a T7 RNA polymerase, respectively; (ii) a T3 promoter or its transcriptional activity variant and a T3 RNA polymerase, respectively; and (iii) an SP6 promoter or its transcriptional activity variant and an SP6 RNA polymerase, respectively. In some embodiments, the second sequence-specific RNA polymerase promoter is a T7 promoter or its transcriptional activity variant, and the second sequence-specific RNA polymerase is a T7 RNA polymerase. In some embodiments, the second sequence-specific RNA polymerase promoter is a bacterial promoter or a transcriptionally active variant thereof, and the second sequence-specific RNA polymerase is a bacterial RNA polymerase.

[0013] In some embodiments, the second sequence-specific RNA polymerase promoter is a eukaryotic promoter or a transcriptionally active variant thereof, and the second sequence-specific RNA polymerase is a eukaryotic RNA polymerase. In some embodiments, the second sequence-specific RNA polymerase promoter is a viral promoter or a transcriptionally active variant thereof, and the second sequence-specific RNA polymerase is a viral RNA polymerase. In some embodiments, the second sequence-specific RNA polymerase promoter is a synthetic promoter, and the second sequence-specific RNA polymerase is a synthetic RNA polymerase. In some embodiments, the first and second promoters and RNA polymerases are the same. In some embodiments, the first and second promoters and RNA polymerases are different. In some embodiments, the target nucleic acid sequence and the second target nucleic acid sequence are adjacent to a polynucleotide encoding an exogenous protein. In some embodiments, the target nucleic acid sequence and the second target nucleic acid sequence are introduced on the same nucleic acid. In some embodiments, the target nucleic acid sequence and another target nucleic acid sequence are introduced on different nucleic acids. In some embodiments, the DNA molecule comprises three or more sequence-specific RNA polymerase promoters, each configured to drive the transcription of different nucleic acid sequences of interest in the presence of a specific sequence-specific RNA polymerase.

[0014] In some embodiments, the DNA molecule further comprises a first padlock binding sequence and a second padlock binding sequence, the first and second padlock binding sequences adjacent to a region containing the target nucleic acid sequence. In some embodiments, the DNA molecule further comprises a third padlock binding sequence and a fourth padlock binding sequence, the third and fourth padlock binding sequences adjacent to a region containing the target second nucleic acid sequence. In some embodiments, prior to in situ sequencing, step (c) further includes (i) contacting cDNA with a first padlock probe having a 5' end and a 3' end, wherein the first padlock probe includes a 5' nucleic acid sequence inversely complementary to a first padlock binding site and a 3' nucleic acid sequence inversely complementary to a second padlock binding site, so that the 5' and 3' nucleic acid sequences can hybridize to the cDNA; (ii) extending the 3' end of the first padlock probe via the nucleic acid sequence of interest using DNA polymerase; (iii) generating a circular DNA template containing the nucleic acid sequence inversely complementary to the nucleic acid sequence of interest by ligating the 5' end of the padlock probe to the extended 3' end of the padlock probe; and (iv) generating additional copies of the nucleic acid sequence of interest using rolling circle amplification of the DNA template. In some embodiments, step (i) further comprises contacting the cDNA with a second padlock probe including a 5' end and a 3' end, the second padlock probe comprising a 5' nucleic acid sequence inversely complementary to a third padlock binding site and a 3' nucleic acid sequence inversely complementary to a fourth padlock binding site, and step (ii) further comprises extending the 3' end of the second padlock probe via the desired second nucleic acid sequence using DNA polymerase. In some embodiments, the first and second padlock binding sequences are different from the third and fourth padlock binding sequences. In some embodiments, the first and second padlock binding sequences are identical to the third and fourth padlock binding sequences. In some embodiments, in situ sequencing is performed directly on the cDNA.

[0015] In some embodiments, the biological sample comprises one or more immune cells. In some embodiments, the one or more immune cells are T cells, NK cells, B cells, mast cells, dendritic cells, macrophages, neutrophils, basophils, and / or eosinophils. In some embodiments, the biological sample comprises a mixture of cells from different species. In some embodiments, the biological sample comprises human cells and mouse cells. In some embodiments, the biological sample comprises human immune cells and mouse cells. In some embodiments, one or more cells in the biological sample consist of cells from a single species. In some embodiments, one or more cells in the biological sample consist of human cells. In some embodiments, the biological sample comprises either or both cancer cells and fibroblasts. In some embodiments, the biological sample comprises one or more human cancer cells. In some embodiments, the biological sample comprises one or more mouse cancer cells. In some embodiments, the biological sample comprises one or more nervous system cells. In some embodiments, the nervous system cells comprise one or more neurons, astrocytes, and microglia.

[0016] In some embodiments, less than 100% of the cells in the biological sample contain the nucleic acid sequence of the target. In some embodiments, the biological sample includes an FFPE sample, and less than 100% of the cells in the biological sample contain the nucleic acid sequence of the target. In some embodiments, all or substantially all of the cells in the biological sample contain the nucleic acid sequence of the target.

[0017] This specification also discloses, in various embodiments, a method for determining in situ the presence, quantity, and / or localization of a target nucleic acid sequence in one or more fixed mammalian cells in a biological sample, the method comprising (a) reverse transcribing a target RNA containing the target nucleic acid sequence using DNA primers in one or more fixed mammalian cells to generate a first cDNA molecule containing the target nucleic acid sequence, wherein the DNA primers include (i) a 5' nucleic acid sequence containing a sequence-specific RNA polymerase promoter, and (ii) a 3' nucleic acid sequence complementary to a portion of the target RNA adjacent to the target nucleic acid sequence. (b) a DNA primer hybridizes to a target RNA, and a first cDNA molecule is generated, comprising a sequence-specific RNA polymerase promoter operably linked to the nucleic acid sequence of interest; (c) the first cDNA molecule is reacted with a sequence-specific RNA polymerase to generate an RNA transcript containing the nucleic acid sequence of interest; (d) the RNA transcript is reacted with a reverse transcriptase to generate a second cDNA molecule containing the nucleic acid sequence of interest; and (d) the second cDNA molecule is sequenced in situ to visualize the nucleic acid sequence of interest in one or more fixed mammalian cells.

[0018] In some embodiments, the method further includes converting the first cDNA molecule to double-stranded DNA using second-strand synthesis prior to step (b). In some embodiments, the 5' nucleic acid sequence of the DNA primer containing a sequence-specific RNA polymerase promoter is dsDNA. In some embodiments, the dsDNA is hybridized dsDNA or hairpin. In some embodiments, the method includes contacting immobilized mammalian cells with RNase to degrade endogenous RNA molecules prior to step (b).

[0019] In some embodiments, the nucleic acid sequence of interest is an exogenous nucleic acid sequence that is introduced into or derived from one or more mammalian cells prior to immobilization. In some embodiments, the nucleic acid sequence of interest is a barcoded polynucleotide. In some embodiments, the exogenous nucleic acid sequence is integrated into the genome of one or more mammalian cells by viral transduction, site-specific nuclease, or site-specific recombinase. In some embodiments, the exogenous nucleic acid sequence is introduced into mammalian cells using a viral vector selected from a lentiviral vector, a retroviral vector, an adenoviral vector, a HSV vector, a baculoviral vector, virus-like particles, pseudotyped virus-like capsids, or an AAV vector. In some embodiments, the exogenous nucleic acid sequence is integrated at a preselected location within the genome. In some embodiments, the exogenous nucleic acid sequence is integrated at a random location within the genome. In some embodiments, the exogenous nucleic acid is not integrated into the mammalian chromosome. In some embodiments, the exogenous nucleic acid is retained within the nucleus of one or more cells. In some embodiments, the exogenous nucleic acid is contained within a plasmid or artificial chromosome.

[0020] In some embodiments, the nucleic acid sequence of interest is an endogenous sequence that varies between cells within a biological sample. In some embodiments, the endogenous nucleic acid sequence encodes a region that includes a T cell receptor, a B cell receptor, an immunoglobulin sequence, a repetitive sequence, or somatic mutations. In some embodiments, the nucleic acid sequence of interest is an endogenous sequence that does not vary between cells within a biological sample.

[0021] In some embodiments, the RNA molecule is mRNA. In some embodiments, the RNA molecule is non-coding RNA. In some embodiments, the RNA molecule includes gRNA. In some embodiments, in situ sequencing is sequencing by synthesis, sequencing by ligation, or sequencing by avidity. In some embodiments, in situ sequencing is sequencing by synthesis. In some embodiments, the nucleic acid sequence of interest is less than 100 nucleotides long, less than 90 nucleotides long, less than 80 nucleotides long, less than 70 nucleotides long, less than 60 nucleotides long, less than 50 nucleotides long, less than 40 nucleotides long, less than 30 nucleotides long, less than 25 nucleotides long, less than 20 nucleotides long, less than 15 nucleotides long, less than 10 nucleotides long, or less than 5 nucleotides long.

[0022] In some embodiments, the promoter is a phage promoter or a transcriptional variant thereof, and the sequence-specific RNA polymerase is a phage RNA polymerase. In some embodiments, the promoter and the sequence-specific RNA polymerase are selected from the group consisting of (i) a T7 promoter or a transcriptional variant thereof and a T7 RNA polymerase, respectively; (ii) a T3 promoter or a transcriptional variant thereof and a T3 RNA polymerase, respectively; and (iii) an SP6 promoter or a transcriptional variant thereof and an SP6 RNA polymerase, respectively. In some embodiments, the promoter is a T7 promoter or a transcriptional variant thereof, and the RNA polymerase is a T7 RNA polymerase.

[0023] In some embodiments, the sequence-specific RNA polymerase promoter is a bacterial promoter, or a transcriptionally active variant thereof, and the sequence-specific RNA polymerase is a bacterial RNA polymerase. In some embodiments, the sequence-specific RNA polymerase promoter is a eukaryotic promoter, or a transcriptionally active variant thereof, and the sequence-specific RNA polymerase is a eukaryotic RNA polymerase. In some embodiments, the sequence-specific RNA polymerase promoter is a viral promoter, or a transcriptionally active variant thereof, and the sequence-specific RNA polymerase is a viral RNA polymerase. In some embodiments, the sequence-specific RNA polymerase promoter is a synthetic promoter, and the sequence-specific RNA polymerase is a synthetic RNA polymerase.

[0024] In some embodiments, the biological sample is fixed using a solution containing formaldehyde and / or paraformaldehyde. In some embodiments, the solution contains 4% paraformaldehyde. In some embodiments, the biological sample includes a formalin-fixed paraffin-embedded (FFPE) sample containing one or more mammalian cells. In some embodiments, the biological sample is fixed by cryo-fixation. In some embodiments, the sample contains an optimal cutting temperature compound, a hydrogel matrix, or a swellable polymeric hydrogel. In some embodiments, the sample is fixed using a solution containing alcohol. In some embodiments, the alcohol is methanol or ethanol. In some embodiments, the sample is fixed using a solution containing glutaraldehyde.

[0025] In some embodiments, the target RNA encodes an exogenous protein, an endogenous protein, or a mixture of exogenous and endogenous proteins. In some embodiments, the target RNA encodes an exogenous protein. In some embodiments, the exogenous protein is a synthetic protein and / or a chimeric protein. In some embodiments, the exogenous protein is independently selected from the group consisting of chimeric antigen receptors (CARs), antibodies, T cell receptors, cytokines, cell surface receptors, transcription factors, signaling proteins, and proteases. In some embodiments, the expression of the exogenous protein is controlled by an endogenous protein in one or more mammalian cells. In some embodiments, the target RNA encodes a nucleic acid sequence that modifies the expression, function, and / or sequence of one or more genes, and is selected from the group consisting of sgRNA, gRNA, shRNA, and miRNA. In some embodiments, the target RNA further comprises a first padlock binding sequence and a second padlock binding sequence, the first and second padlock binding sequences being adjacent to the nucleic acid sequence of interest. In some embodiments, before performing in situ sequencing, step (d) further includes (i) contacting a second cDNA molecule with a padlock probe having a 5' end and a 3' end, wherein the padlock probe includes a 5' nucleic acid sequence inversely complementary to a first padlock binding site and a 3' nucleic acid sequence inversely complementary to a second padlock binding site, so that the 5' and 3' nucleic acid sequences of the padlock probe can hybridize to the cDNA; (ii) extending the 3' end of the padlock probe using DNA polymerase; (iii) generating a circular DNA template containing a nucleic acid sequence inversely complementary to the nucleic acid sequence of interest by ligating the 5' end of the padlock probe to the extended 3' end of the padlock probe; and (iv) generating additional copies of the nucleic acid sequence of interest using rolling circle amplification of the DNA template.

[0026] In some embodiments, the biological sample comprises one or more immune cells. In some embodiments, the one or more immune cells are T cells, NK cells, B cells, mast cells, dendritic cells, macrophages, neutrophils, basophils, and / or eosinophils. In some embodiments, the biological sample comprises a mixture of cells from different species. In some embodiments, the biological sample comprises human cells and mouse cells. In some embodiments, the biological sample comprises human immune cells and mouse cells. In some embodiments, one or more cells in the biological sample consist of cells from a single species. In some embodiments, one or more cells in the biological sample consist of human cells.

[0027] In some embodiments, less than 100% of the cells in the biological sample contain the nucleic acid sequence of the target. In some embodiments, the biological sample includes an FFPE sample, and less than 100% of the cells in the biological sample contain the nucleic acid sequence of the target. In some embodiments, all or substantially all of the cells in the biological sample contain the nucleic acid sequence of the target.

[0028] In some embodiments, the method further includes detecting the presence, quantity, and / or localization of one or more additional analytes within a cell. In some embodiments, the one or more additional analytes are independently selected from the group consisting of proteins, RNA, DNA stained in a non-sequence-specific manner, DNA having a specific sequence, DNA mutations, lipids including but not limited to phospholipids and sphingolipids, carbohydrates including but not limited to monosaccharides and polysaccharides, metabolites, small molecules, cellular structures, and tissue structures. In some embodiments, the method further includes detecting the presence, quantity, and / or localization of one or more protein analytes within a cell. In some embodiments, the one or more protein analytes are detected by immunofluorescence microscopy. In some embodiments, the method further includes detecting the presence, quantity, and / or localization of one or more RNA analytes within a cell. In some embodiments, the one or more RNA analytes are detected by hybridization chain reaction (HCR).

[0029] Furthermore, in various embodiments, kits are disclosed herein that include (a) a sequence-specific RNA polymerase, (b) a reverse transcriptase, (c) a reagent for in situ sequencing, and (d) instructions for using components (a) to (c) to determine in situ the presence, quantity, and / or localization of a nucleic acid sequence of interest in one or more fixed mammalian cells in a biological sample. In some embodiments, (i) in situ sequencing is sequencing by synthesis, and the reagent for in situ sequencing comprises (A) a plurality of detectably labeled nucleotides and (B) DNA polymerase; (ii) in situ sequencing is sequencing by ligation, and the reagent for in situ sequencing comprises (A) a plurality of detectably labeled oligonucleotides comprising degenerate bases and (B) DNA ligase; or (iii) in situ sequencing is sequencing by avidity, and the reagent for in situ sequencing comprises (A) a plurality of detectably labeled avidites and (B) manipulated DNA polymerase.

[0030] In some embodiments, the kit further includes (i) a DNA primer comprising (A) a 3' nucleic acid sequence complementary to a portion of the target RNA adjacent to the nucleic acid sequence of interest, and (B) a 5' nucleic acid sequence containing a sequence-specific RNA polymerase promoter, or further instructions for designing the DNA primer, and (ii) further instructions for reverse transcribing the target RNA using the DNA primer to generate a cDNA molecule. In some embodiments, the 5' nucleic acid sequence of the DNA primer containing the sequence-specific RNA polymerase promoter is dsDNA. In some embodiments, the dsDNA is hybridized dsDNA or hairpin. In some embodiments, the kit further includes (i) reagents for performing second-strand synthesis on the cDNA molecule, and / or (ii) further instructions for performing second-strand synthesis.

[0031] Furthermore, in various embodiments herein, a population of manipulated cells is disclosed that includes an exogenous promoter juxtaposed with an endogenous genomic region containing a genomic sequence that is variable among cells in the population, the exogenous promoter being able to drive the expression of the genomic sequence that is variable among cells in the population, and the exogenous promoter is inserted in a site-specific manner. In some embodiments, the exogenous promoter is selected from the group consisting of the T7 promoter, the T3 promoter, and the SP6 promoter. In some embodiments, the promoter is the T7 promoter. In some embodiments, the exogenous promoter and the genomic sequence that is variable among cells in the population are separated by approximately 2 kilobases (kb) or less, 1.5 kb or less, 1 kb or less, 900 (base pairs) bp or less, 800 bp or less, 700 bp or less, 600 bp or less, 500 bp or less, 400 bp or less, 300 bp or less, 250 bp or less, 200 bp or less, 150 bp or less, 100 bp or less, 50 bp or less, or 0 bp. In some embodiments, the separation is located within the genomic DNA sequence, exon-coding sequence, or RNA sequence of the cell.

[0032] In some embodiments, the manipulated cells are mammalian cells. In some embodiments, the mammalian cells are human cells. In some embodiments, the manipulated cells are immune cells. In some embodiments, the immune cells are T cells, NK cells, B cells, mast cells, dendritic cells, macrophages, neutrophils, basophils, and / or eosinophils. In some embodiments, the genomic sequence, which is variable between cells within a population, encodes a region containing T cell receptors, B cell receptors, immunoglobulin sequences, repetitive sequences, or somatic mutations.

[0033] This specification also discloses, in various embodiments, a method for determining in situ the presence, quantity, and / or localization of a nucleic acid sequence of interest in one or more mammalian cells within a biological sample, the method comprising: (a) introducing an exogenous DNA molecule containing the nucleic acid sequence of interest operably linked to a sequence-specific RNA polymerase promoter into one or more mammalian cells; (b) immobilizing the mammalian cells; (c) reacting the exogenous DNA molecule with a sequence-specific RNA polymerase to produce an RNA transcript of the nucleic acid sequence of interest; (d) reacting the RNA transcript with reverse transcriptase in situ to produce a cDNA molecule containing the nucleic acid sequence of interest; and (e) sequencing the cDNA molecule in situ to visualize copies of the nucleic acid sequence of interest in one or more immobilized mammalian cells. In some embodiments, the exogenous DNA molecule is genetically engineered within the genome of one or more mammalian cells. In some embodiments, the exogenous DNA molecule is genetically engineered upstream of one or more loci of interest.

[0034] This specification also discloses, in various embodiments, a method for determining in situ the presence, quantity, and / or localization of a target nucleic acid sequence in one or more mammalian cells within a biological sample, the method comprising (a) immobilizing mammalian cells, and (b) introducing DNA primers into one or more mammalian cells, the DNA primers comprising (i) a 5' nucleic acid sequence containing a sequence-specific RNA polymerase promoter, and (ii) a 3' nucleic acid sequence complementary to a portion of the target RNA adjacent to the target nucleic acid sequence, wherein the DNA primers hybridize to the target RNA containing the target nucleic acid sequence. The method comprises (c) reverse transcribing a target RNA using DNA primers to generate a first cDNA molecule containing a target nucleic acid sequence operably linked to the target nucleic acid sequence; (d) reacting the first cDNA molecule with a sequence-specific RNA polymerase to generate an RNA transcript containing the target nucleic acid sequence; (e) reacting the RNA transcript with a reverse transcriptase to generate a second cDNA molecule containing the target nucleic acid sequence; and (f) sequencing the second cDNA molecule in situ to visualize the target nucleic acid sequence in one or more fixed mammalian cells.

[0035] These and other aspects and features of the present invention are described in the following detailed description and claims.

[0036] The present invention can be better understood by referring to the following drawings. Note that, where applicable, similar or identical reference numerals may be used in the drawings to indicate similar or identical functions. [Brief explanation of the drawing]

[0037] [Figure 1] Figures A and B are schematic diagrams of exemplary methods provided herein according to embodiments. Figure A illustrates an exemplary method in which the transcription of a nucleic acid barcode is driven using a T7 promoter and T7 RNA polymerase in fixed cells, and then visualized by subsequent reverse transcription and synthesis sequencing. Figure B illustrates a schematic diagram in which a target nucleic acid sequence is amplified using a sequence-specific RNA polymerase in fixed cells, and then visualized by reverse transcription and synthesis sequencing. [Figure 2A] Figure 2A shows a schematic diagram of an exemplary method provided herein, according to an embodiment. Figure 2A illustrates a schematic diagram of an exemplary method in which an exogenous T7 promoter is introduced into a nucleic acid barcode in a fixed cell, which is then amplified using T7 RNA polymerase and subsequently visualized by double-strand synthesis, reverse transcription, and sequencing. [Figure 2B] Figure 2B shows a schematic diagram of an exemplary method provided herein, according to an embodiment. Figure 2B illustrates a schematic diagram of an exemplary method in which an exogenous T7 promoter is introduced into a nucleic acid barcode in a fixed cell using a primer containing a double-stranded portion with the T7 promoter. The nucleic acid barcode is then amplified using T7 RNA polymerase and subsequently visualized by sequencing by reverse transcription and synthesis. [Figure 2C]Figure 2C shows a schematic diagram of an exemplary method provided herein, according to an embodiment. Figure 2C illustrates the synthesis of a cDNA molecule containing an exogenous promoter operably ligated to a target nucleic acid sequence by introducing an exogenous promoter and a primer containing a nucleic acid sequence partially overlapping with the target nucleic acid sequence. [Figure 3] This figure shows microscopic images of cultured cells that have undergone sequencing by reverse transcription and in-situ synthesis, with or without the initial step of barcode amplification using T7 RNA polymerase (lower panel). [Figure 4] This figure shows microscopic images of formalin-fixed paraffin-embedded (FFPE) tissue sections that underwent reverse transcription and sequencing by in-situ synthesis, with or without the initial step of barcode amplification using T7 RNA polymerase (lower panel). [Figure 5] This figure shows microscopic images of formalin-fixed paraffin-embedded (FFPE) tissue sections that have undergone sequencing by reverse transcription and in-situ synthesis, with an initial step of barcode amplification using T7 polymerase for 3 hours (upper panel) or 18 hours (lower panel). [Figure 6] This figure shows microscopic images of formalin-fixed, paraffin-embedded (FFPE) tissue sections that underwent barcode amplification with T7 RNA polymerase, followed by reverse transcription and sequencing by in-situ synthesis. The panels show tissue that underwent initial RNase treatment to degrade native mRNA before reverse transcription (lower panel) or tissue that did not (upper panel). [Figure 7] This figure shows microscopic images of formalin-fixed, paraffin-embedded (FFPE) tissue sections that underwent barcode amplification with T7 RNA polymerase, followed by reverse transcription and sequencing by synthesis in situ. The panel illustrates tissues that have undergone sequencing by synthesis for the indicated number of rounds. [Figure 8] This is a schematic diagram of an exemplary workflow in which a variable barcode sequence is amplified using a promoter embedded in a biological sample, followed by amplification of the cDNA product. [Figure 9A] This figure shows microscopic images of FFPE tissue sections from a mouse model of orthotopic gastric patient xenograft tumor using seven unique CAR designs, after barcode amplification, reverse transcription, cDNA amplification, and sequencing by in-situ synthesis (SBS) using T7 RNA polymerase. Figure 9A illustrates the DAPI signal from the entire tumor using a 1 mm scale bar. [Figure 9B] This figure shows microscopic images of FFPE tissue sections from an orthotopic gastric patient xenograft tumor mouse model using seven unique CAR designs, after barcode amplification, reverse transcription, cDNA amplification, and sequencing by synthesis (SBS) in situ using T7 RNA polymerase. Figure 9B shows the tissue containing the area enclosed by the rectangle in Figure 9A, with the DAPI signal superimposed on the SBS signal from all nucleotides (A, G, C, T) in round 1, illustrated with a 100 μm scale bar. [Figure 9C] This figure shows microscopic images of FFPE tissue sections from an orthotopic gastric patient-derived xenograft tumor mouse model using seven unique CAR designs, after barcode amplification, reverse transcription, cDNA amplification, and sequencing by synthesis in situ (SBS) using T7 RNA polymerase. Figure 9C shows the tissue containing the area enclosed by the rectangle in Figure 9B, illustrating barcode reading across multiple rounds using signals from each fluorescence channel in each round of SBS. The dashed lines indicate three individual cells within this field of view, each with a different amplified barcode, which corresponds to one of the seven barcodes in the library. Barcoded cells are shown as dashed lines within the DAPI panel for all rounds, but only within the SBS panel corresponding to the barcode signal for each SBS round. All subpanels have a scale bar of 20 μm. [Figure 10A]This figure shows microscopic images of FFPE tissue sections from a HepG2 cell line-derived xenograft tumor mouse model after sequencing by barcode amplification, reverse transcription, cDNA amplification, and in-situ synthesis using T7 RNA polymerase, with nine unique CAR designs. Figure 10A illustrates the DAPI signal from the entire tumor using a 2 mm scale bar. [Figure 10B] This figure shows microscopic images of FFPE tissue sections from a HepG2 cell line-derived xenograft tumor mouse model, following barcode amplification, reverse transcription, cDNA amplification, and sequencing by in-situ synthesis using T7 RNA polymerase, with nine unique CAR designs. Figure 10B shows the tissue containing the area enclosed by the rectangle in Figure 10A, with the DAPI signal superimposed on the SBS signal from all nucleotides (A, C, G, T) in Round 1, illustrated with a 75 μm scale bar. [Figure 10C] This figure shows microscopic images of FFPE tissue sections from a HepG2 cell line-derived xenograft tumor mouse model after barcode amplification, reverse transcription, cDNA amplification, and sequencing by in-situ synthesis using T7 RNA polymerase. Figure 10C shows the tissue containing the area enclosed by the rectangle in Figure 10B, illustrating barcode reading across multiple rounds using signals from each fluorescence channel in each round of SBS. The dashed lines indicate four individual cells within this field of view, each with a different amplified barcode, which corresponds to one of the nine barcodes in the library. Barcoded cells are shown as dashed lines within the DAPI panel for all rounds, but only within the SBS channel corresponding to the barcode signal for each SBS round. All subpanels have a scale bar of 20 μm. [Figure 11A]This figure shows microscopic images of FFPE tissue sections from a Hep G2 cell line-derived xenograft tumor mouse model after sequencing by barcode amplification, reverse transcription, cDNA amplification, and in-situ synthesis using T7 RNA polymerase, with 56 unique CAR designs (including armored and unarmored CAR constructs). Figure 11A illustrates the DAPI signal from the entire tumor using a 2 mm scale bar. [Figure 11B] This figure shows microscopic images of FFPE tissue sections from a Hep G2 cell line-derived xenograft tumor mouse model after sequencing by barcode amplification, reverse transcription, cDNA amplification, and in-situ synthesis using T7 RNA polymerase, with 56 unique CAR designs (including armored and unarmored CAR constructs). Figure 11B shows the tissue, including the area enclosed by the rectangle in panel A, with the DAPI signal superimposed on the SBS signal from all nucleotides (A, C, G, T) in round 1, illustrated with a 100 μm scale bar. [Figure 11C] This figure shows microscopic images of FFPE tissue sections from a Hep G2 cell line-derived xenograft tumor mouse model after sequencing by barcode amplification, reverse transcription, cDNA amplification, and in-situ synthesis using T7 RNA polymerase, with 56 unique CAR designs (including armored and unarmored CAR constructs). Figure 11C shows the tissue containing the area enclosed by the square in panel B, illustrating barcode reading across multiple rounds using signals from each fluorescence channel in each round of SBS. The dashed lines indicate three individual cells within this field of view, each with a different amplified barcode, which corresponds to one of the 56 barcodes in the library. Barcoded cells are shown as dashed lines within the DAPI panel for all rounds, but only within the SBS panel corresponding to the barcode signal for each SBS round. All subpanels have a scale bar of 20 μm. [Figure 12A]This figure shows microscopic images of FFPE tissue sections from an AsPC-1 cell line-derived xenograft tumor mouse model after sequencing by barcode amplification, reverse transcription, cDNA amplification, and in-situ synthesis using T7 RNA polymerase, with 80 unique CAR designs (including armored and unarmored CAR constructs). Figure 12A illustrates the DAPI signal from the entire tumor using a 1 mm scale bar. [Figure 12B] This figure shows microscopic images of FFPE tissue sections from an AsPC-1 cell line xenograft tumor mouse model after sequencing by barcode amplification, reverse transcription, cDNA amplification, and in-situ synthesis using T7 RNA polymerase, with 80 unique CAR designs (including armored and unarmored CAR constructs). Figure 12B shows the tissue containing the area enclosed by the rectangle in Figure 12A, with the DAPI signal superimposed on the SBS signal from all nucleotides (A, C, G, T) in round 1, illustrated with a 100 μm scale bar. [Figure 12C] This figure shows microscopic images of FFPE tissue sections from an AsPC-1 cell line xenograft tumor mouse model after sequencing by barcode amplification, reverse transcription, cDNA amplification, and in-situ synthesis using T7 RNA polymerase, with 80 unique CAR designs (including armored and unarmored CAR constructs). Figure 12C shows the tissue containing the area enclosed by the rectangle in Figure 12B, illustrating barcode reading across multiple rounds using signals from each fluorescence channel in each round of SBS. The dashed lines indicate two individual cells within this field of view, each with a different amplified barcode, corresponding to one of the 80 barcodes in the library. Barcoded cells are shown as dashed lines within the DAPI panel for all rounds, but only within the SBS panel corresponding to the barcode signal for each SBS round. All subpanels have a scale bar of 20 μm. [Figure 13A]This figure shows microscopic images of FFPE tissue sections from an AsPC-1 cell line-derived xenograft tumor mouse model, following sequencing by barcode amplification, reverse transcription, cDNA amplification, and in-situ synthesis using T7 RNA polymerase, using 10 unique CAR and shRNA designs. Figure 13A illustrates the DAPI signal from the entire tumor using a 1 mm scale bar. [Figure 13B] This figure shows microscopic images of FFPE tissue sections from an AsPC-1 cell line-derived xenograft tumor mouse model, following sequencing by barcode amplification, reverse transcription, cDNA amplification, and in-situ synthesis using T7 RNA polymerase, using 10 unique CAR and shRNA designs. Figure 13B shows the tissue containing the area enclosed by the rectangle in Figure 13A, with the DAPI signal superimposed on the SBS signal from all nucleotides (A, C, G, T) in Round 1, illustrated with a 100 μm scale bar. [Figure 13C] This figure shows microscopic images of FFPE tissue sections from an AsPC-1 cell line-derived xenograft tumor mouse model after sequencing by barcode amplification, reverse transcription, cDNA amplification, and in-situ synthesis using T7 RNA polymerase, with 10 unique CAR and shRNA designs. Figure 13C shows the tissue containing the area enclosed by the rectangle in Figure 13B, illustrating barcode reading across multiple rounds using signals from each fluorescence channel in each round of SBS. The dashed lines indicate three individual cells within this field of view, each with a different amplified barcode, corresponding to one of the 10 barcodes in the library. Barcoded cells are shown as dashed lines within the DAPI panel for all rounds, but only within the SBS channel corresponding to the barcode signal for each SBS round. All subpanels have a scale bar of 20 μm. [Figure 14A]This figure shows the quantification of barcodes detected in vivo from transduced T cells using the nine barcoded CAR construct libraries shown in Table 1. The x-axis represents the number of barcoded CAR construct designs, and the y-axis represents the number of each design detected. The panel illustrates barcodes detected in FFPE tissue sections containing CAR T cells prepared from T cell donor 1. Barcode holdout (BH) designs that were not part of the CAR library were not detected very often, but barcodes included in the nine CAR libraries were detected at high levels. [Figure 14B] This figure shows the quantification of barcodes detected in vivo from transduced T cells using the nine barcoded CAR construct libraries shown in Table 1. The x-axis represents the number of barcoded CAR construct designs, and the y-axis represents the number of each design detected. The panel illustrates barcodes detected in FFPE tissue sections containing CAR T cells prepared from T cell donor 2. Barcode holdout (BH) designs that were not part of the CAR library were not detected very often, but barcodes included in the nine CAR libraries were detected at high levels. [Figure 14C] This figure shows the quantification of barcodes detected in vivo from transduced T cells using the nine barcoded CAR construct libraries shown in Table 1. The x-axis represents the number of barcoded CAR construct designs, and the y-axis represents the number of each design detected. The panel illustrates barcodes detected in FFPE tissue sections containing CAR T cells prepared from T cell donor 3. Barcode holdout (BH) designs that were not part of the CAR library were not detected very often, but barcodes included in the nine CAR libraries were detected at high levels. [Figure 15] This figure shows the quantification of barcodes detected in vivo from transduced T cells using a library of seven barcoded CAR constructs shown in Table 2. The x-axis represents the number of barcoded designs, and the y-axis represents the number of each design detected in FFPE tissue sections of orthotopic patient-derived xenograft tumors. All seven barcoded designs were detected. [Figure 16A] This figure shows the quantification of barcodes detected from transduced T cells using the 56 barcoded CAR construct libraries shown in Table 3. The barcoded CAR designs are designated as D-1 to D-56 (true barcodes), and the barcode holdouts are designated as BH-1 to BH-13. Figure 16A shows the number of designs on the x-axis and the number of each design detected in FFPE tissue sections from xenograft tumors derived from Hep G2 cell lines on the y-axis. Barcodes from the 56 CAR design libraries were detected at higher levels than the barcode holdouts. [Figure 16B] This figure shows the quantification of barcodes detected from transduced T cells using the 56 barcoded CAR construct libraries shown in Table 3. The barcoded CAR designs are designated as D-1 to D-56 (true barcodes), and the barcode holdouts are designated as BH-1 to BH-13. Figure 16B illustrates the total raw count of all detected true barcodes for all barcode holdouts detected in tissue sections of Panel A. [Figure 17A] This figure shows the quantification of barcodes detected in vivo from transduced T cells using a library of 80 barcoded CAR design constructs shown in Table 4. The number of designs is shown on the x-axis, and the number of barcodes detected for each design is shown on the y-axis. The panel shows barcodes detected in FFPE tissue sections from xenografts derived from the AsPC-1 cell line, containing library CAR T cells prepared from T cell donor 1. [Figure 17B] This figure shows the quantification of barcodes detected in vivo from transduced T cells using a library of 80 barcoded CAR design constructs shown in Table 4. The number of designs is shown on the x-axis, and the number of detected barcodes for each design is shown on the y-axis. The panel shows barcodes detected in FFPE tissue sections from AsPC-1 cell line-derived xenografts containing library CAR T cells prepared from T cell donor 2. [Figure 18A]Figures A and B show microscopic images of in vitro T cells transduced with a library of nine barcoded CAR design constructs from Table 1, following barcode amplification, reverse transcription, cDNA amplification, and in-situ SBS using T7 RNA polymerase. A illustrates the DAPI signals from all cells in the field of view with a 100 μm scale bar. B shows the tissue containing the area enclosed by the rectangle in A, illustrating barcode readings across multiple rounds using the signals from each fluorescence channel in each round of SBS. The dashed lines indicate four individual cells in this field of view, each with a different amplified barcode, which corresponds to one of the nine barcodes in the library. Barcoded cells are shown as dashed lines within the DAPI panel for all rounds, but only within the SBS channel corresponding to the barcode signal for each SBS round. The scale bar in all subpanels is 20 μm. [Figure 18B] Same as above. [Figure 19] Figures A and B show microscopic images of in vitro T cells transduced with a library of 16 CAR design constructs, each having a unique barcode following a T7 promoter, following barcode amplification, reverse transcription, cDNA amplification, and in-situ SBS using T7 RNA polymerase (each cell contains 0, 1, 2, or more constructs). A illustrates the DAPI signals from all cells in the field of view with a 100 μm scale bar. B shows the tissue containing the area enclosed by the square in A, illustrating barcode readings across multiple rounds using the signals from each fluorescent channel in each round of SBS. The square dashed line shape indicates a single cell with one distinct amplified barcode, while the diamond and circular dashed line shapes indicate a single cell with two distinct amplified barcodes. Barcoded cells are shown as dashed lines within the DAPI panel for all rounds, but only within the SBS channel corresponding to the barcode signal for each SBS round. The scale bar for all subpanels is 20 μm. [Figure 20] This figure shows the quantification of barcodes detected in vitro from transduced T cells using the library of nine barcoded CAR design constructs shown in Table 1. The x-axis represents the number of construct designs, and the y-axis represents the number of barcodes detected for each design. [Figure 21] This figure shows the quantification of barcodes detected in vitro from transduced T cells using the library of seven barcoded CAR design constructs shown in Table 2. The x-axis represents the number of construct designs, and the y-axis represents the number of each design detected. [Figure 22] Figures A and B show the quantification of barcodes detected in vivo from transduced T cells using a library of nine barcoded CAR constructs shown in Table 1. A represents the raw count of barcodes, and B illustrates the barcode ratio. Donor numbers indicate barcodes detected in FFPE tissue sections containing CAR T cells prepared from each of the three donors. True barcodes include CAR design numbers 1–8 for all three donors, as well as design 9A for donors 1 and 2, and design 9B for donor 3. Barcode holdouts include 60 negative control barcodes, BH numbers 1–59 for all three donors, as well as design 9B for donors 1 and 2, and design 9A for donor 3. [Figure 23] This is a schematic diagram comparing in vivo barcode amplification using either the SP6 or T7 promoter. [Figure 24A]Figures A-F show microscopic images of FFPE-embedded tissue sections from an orthotopic patient-derived xenograft tumor mouse model using seven CAR constructs (each with a unique barcode) after barcode amplification, reverse transcription, cDNA amplification, and sequencing by synthesis (SBS) using a specified sequence-specific RNA polymerase. The CAR library contained six constructs with a barcode after the T7 promoter and one construct with a barcode after both the T7 and SP6 promoters. A and D illustrate the DAPI signal from the entire tumor section with a 1 mm scale bar. B and E show the tissue containing the regions enclosed in squares in A and D, respectively, with the DAPI signal overlaid with the SBS signal from all nucleotides (A, C, G, T) in round 1, with a 100 μm scale bar. C and F show the tissue containing the regions enclosed in squares in panels B and E, respectively, and illustrate barcode readings across multiple rounds using the signal from each fluorescence channel in each round of SBS. The dashed lines indicate individual cells with different amplified barcodes within these fields of view. The shape in C illustrates multiple barcodes from multiple constructs with the T7 promoter. The shape in E illustrates a single barcode from a single construct with the SP6 promoter. Barcoded cells are indicated by dashed lines within the DAPI panel for all rounds, except for each SBS round, which is indicated only within the SBS channel corresponding to the barcode signal. The scale bar in all subpanels is 20 μm. [Figure 24B] Same as above. [Figure 24C] Same as above. [Figure 24D] Same as above. [Figure 24E] Same as above. [Figure 24F] Same as above. [Figure 25A] This figure shows microscopic images of FFPE tissue sections from a xenograft tumor mouse model derived from the Hep G2 cell line. Figure 25A illustrates the DAPI signal from the entire tumor using a 2 mm scale bar. [Figure 25B] This figure shows microscopic images of FFPE tissue sections from a xenograft tumor mouse model derived from the Hep G2 cell line. Figure 25B shows the tissue, including the area enclosed by the rectangle in Figure 25A, with the DAPI signal superimposed on the SBS signal from all nucleotides (A, C, G, T) in round 1, illustrated with a 75 μm scale bar. [Figure 25C] This figure shows microscopic images of FFPE tissue sections from a xenograft tumor mouse model derived from the Hep G2 cell line. Figure 25C shows the tissue containing the area enclosed by the rectangle in Figure 25B, illustrating barcode readings across multiple rounds using signals from each fluorescence channel in each round of SBS. The dashed lines indicate two individual cells within this field of view, each with a different amplified barcode, corresponding to one of 56 barcodes in the library. Barcoded cells are shown as dashed lines within the DAPI panel for all rounds, but only within the SBS panel corresponding to the barcode signal for each SBS round. [Figure 25D] This figure shows microscopic images of FFPE tissue sections from a xenograft tumor mouse model derived from the Hep G2 cell line. Figure 25D shows the same tissue region as Figure 25C, with DAPI staining on the left, barcode signals from all channels of Round 1 in the center, and staining images of protein markers including CD8, granzyme B, LAG3, cytokeratin, and PDL1 in the right panel. All subpanels have a scale bar of 20 μm. [Figure 26A] This figure shows microscopic images of FFPE tissue sections from a xenograft tumor mouse model derived from the AsPC-1 cell line. Figure 26A illustrates the DAPI signal from the entire tumor using a 2 mm scale bar. [Figure 26B]This figure shows microscopic images of FFPE tissue sections from a xenograft tumor mouse model derived from the AsPC-1 cell line. Figure 26B shows the tissue, including the area enclosed by the rectangle in panel A, with the DAPI signal superimposed on the SBS signal from all nucleotides (A, C, G, T) in round 1, illustrated with a 75 μm scale bar. [Figure 26C] This figure shows microscopic images of FFPE tissue sections from a xenograft tumor mouse model derived from the AsPC-1 cell line. Figure 26C shows the tissue, including the area enclosed by the square in panel B, and illustrates barcode readings across multiple rounds using signals from each fluorescence channel in each round of SBS. The dashed lines indicate two individual cells within this field of view, each with a different amplified barcode, which correspond to one of 80 barcodes in the library. Barcoded cells are shown as dashed lines within the DAPI panel for all rounds, but only within the SBS panel corresponding to the barcode signal for each SBS round. [Figure 26D] This figure shows microscopic images of FFPE tissue sections from a xenograft tumor mouse model derived from the AsPC-1 cell line. Figure 26D shows the same tissue region as Figure 26C, with DAPI staining on the left, barcode signals from all channels of Round 1 in the center, and staining images of protein markers including CD8, granzyme B, LAG3, and cytokeratin in the right panel. All subpanels have a scale bar of 20 μm. [Figure 27] This graph shows the proportion of barcoded cells that overlap with T cell marker-positive cells (CD45-positive) or T cell marker-negative cells (CD45-negative) in FFPE tissue sections from a xenograft tumor mouse model derived from the AsPC-1 cell line injected with a cell library consisting of 80 barcoded constructs. The x-axis represents the staining status of CD45 cells, and the y-axis represents the proportion of barcodes in the tumor section that were assigned to CD45-positive or CD45-negative cells. [Figure 28A]This figure shows microscopic images of FFPE tissue sections from a xenograft tumor mouse model derived from the Hep G2 cell line. Figure 28A illustrates the DAPI signal from the entire tumor using a 2 mm scale bar. [Figure 28B] This figure shows microscopic images of FFPE tissue sections from a xenograft tumor mouse model derived from the Hep G2 cell line. Figure 28B shows the tissue, including the area enclosed by the rectangle in panel A, with the DAPI signal superimposed on the SBS signal from all nucleotides (A, C, G, T) in round 1, illustrated with a 100 μm scale bar. [Figure 28C] This figure shows microscopic images of FFPE tissue sections from a xenograft tumor mouse model derived from the Hep G2 cell line. Figure 28C shows the tissue after one round of SBS treatment, including the area enclosed by the rectangle in Figure 28B. The tissue was imaged after round 1 of SBS to read the first nucleotide of the barcode in individual cells. The signals from each channel are shown separately to show the fluorescence reading of individual barcodes. The shape of the dashed lines indicates individual cells with a clear SBS signal in round 1. [Figure 28D] This figure shows microscopic images of FFPE tissue sections from a xenograft tumor mouse model derived from the Hep G2 cell line. Figure 28D shows the same tissue region as Figure 28C, with DAPI staining on the left, barcode signals from all channels of Round 1 overlaid with DAPI staining in the center, and CAR RNA staining overlaid with DAPI staining in the right panel. All subpanels have a scale bar of 20 μm. [Modes for carrying out the invention]

[0038] I. Definition The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” refer to biopolymers of nucleotides and, unless otherwise indicated in context, include modified and unmodified nucleotides, both DNA and RNA, and modified nucleic acid backlines. Nucleic acids are said to have a “5' end” and a “3' end” because, typically, mononucleotides react to form oligonucleotides by which the 5' phosphate group or equivalent of one nucleotide optionally binds to the 3' hydroxyl group or equivalent of an adjacent nucleotide via a phosphodiester bond or other preferred bond. The primers and oligonucleotides used in the methods disclosed herein include nucleotides. In some embodiments, the nucleotides include any compound that can selectively bind to a polymerase or can be polymerized by a polymerase, and may include, but are not limited to, any native nucleotide or its analogues. Typically, but not necessarily, after a nucleotide has selectively bound to a polymerase, the polymerase polymerizes the nucleotide into a nucleic acid chain.

[0039] The terms “amplify,” “amplification,” and “amplification reaction,” and similar expressions, generally refer to any action or process that replicates or copies at least a portion of a nucleic acid molecule (referred to as the “template”) to produce at least one additional nucleic acid molecule. The additional nucleic acid molecule optionally contains a sequence that is substantially identical or substantially complementary to at least a substantial portion of the template nucleic acid molecule. The template nucleic acid molecule may be single-stranded or double-stranded, and the additional nucleic acid molecule may independently be single-stranded or double-stranded. In some embodiments, amplification involves an enzyme-catalyzed, template-dependent in vitro reaction for producing at least one copy of at least a substantial portion of a nucleic acid molecule, or at least one copy of a nucleic acid sequence complementary to at least a substantial portion of a nucleic acid molecule. Amplification optionally involves linear or exponential replication of a nucleic acid molecule.

[0040] "Complementarity" or "complementary" refers to the ability of a nucleic acid to form hydrogen bonds (or multiple hydrogen bonds) with another nucleic acid sequence, either in the conventional Watson-Crick format or in other non-conventional formats, i.e., the ability to hybridize with another nucleic acid sequence. As used herein, "hybridization" refers to the binding, double-stranding, or hybridization of a molecule with only a specific nucleotide sequence under low, medium, or high stringent conditions (including when that sequence is present in the DNA or RNA of a complex mixture, e.g., whole cells). See, for example, Ausubel, et al., Current Protocols In Molecular Biology, John Wiley & Sons, New York, NY, 1993. If a nucleotide at a particular position in a polynucleotide can form a Watson-Crick pair with a nucleotide at the same position on an antiparallel DNA or RNA strand, then that polynucleotide and its DNA or RNA molecule are complementary at that position. Two polynucleotides are said to be "reverse complements" of each other if all nucleotides in a polynucleotide can form a Watson-Crick pair with the nucleotide at the corresponding position in an antiparallel polynucleotide.

[0041] The use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including their grammatical equivalents, is generally open-ended and non-restrictive, and should be understood not to exclude any additional unspecified elements or steps unless specifically stated or understood otherwise from the context.

[0042] As used herein, the terms “subject” and “patient” refer to the organism that is the source of the sample to be analyzed by the method described herein. Preferably, such organisms include, but are not limited to, mammals (e.g., mice, monkeys, horses, cattle, pigs, dogs, cats, etc.), and more preferably, humans.

[0043] Where the term “about” is used before a quantitative value, unless otherwise specified, the present invention also encompasses the specific quantitative value itself. As used herein, the term “about” refers to a variation of ±10% from a baseline value, unless otherwise specified or implied. To avoid misunderstanding, where the term “about” is used before a list of numbers, the term “about” applies to each element of that list. For example, “about 1, 2, 3, 4, or 5” should be understood to mean “about 1, about 2, about 3, about 4, or about 5.”

[0044] It should be understood that, as long as the present invention is implementable, the order of the steps or the order in which specific actions are performed is not important. Furthermore, two or more steps or actions can be performed simultaneously.

[0045] Unless specifically requested, any use of examples or illustrative words herein, such as “such as” or “including,” is intended solely to better illustrate the invention and not to limit its scope. Language herein should not be construed as indicating any non-claiming element essential to the practice of the invention.

[0046] In this application, when an element or component is said to be included in and / or selected from a list of enumerated elements or components, it should be understood that the element or component may be any of the enumerated elements or components, or may be selected from a group consisting of two or more of the enumerated elements or components.

[0047] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein, whether expressly or implicitly, can be combined in various ways without departing from the spirit and scope of the invention. For example, where a particular compound is referenced, unless otherwise understood from the context, that compound can be used in various embodiments of the compositions and / or methods of the invention. In other words, while embodiments in this application are described and depicted in such a manner that obvious and concise applications are described and depicted, it is intended and understood that embodiments can be combined or separated in various ways without departing from the teachings and the invention(s) of the invention. For example, it should be understood that all features described and depicted herein are applicable to all embodiments of the invention(s) described and depicted herein.

[0048] II. Method This specification discloses, in various embodiments, methods for detecting target nucleic acids (e.g., nucleic acid barcodes) in a biological sample via imaging.

[0049] This disclosure generally relates to methods and compositions for in situ detection of one or more target nucleic acid sequences (e.g., nucleic acid barcodes) in a biological sample. This specification provides, for example, methods and compositions for detecting multiple different analytes in a sample by imaging. In some embodiments, the group of nucleic acid barcodes is designed such that the minimum mutual Hamming distance is 6. In some embodiments, the group of nucleic acid barcodes is designed such that the minimum mutual Hamming distance is 5. In some embodiments, the group of nucleic acid barcodes is designed such that the minimum mutual Hamming distance is 4. In some embodiments, the group of nucleic acid barcodes is designed such that the minimum mutual Hamming distance is 3. In some embodiments, the group of nucleic acid barcodes is designed such that the minimum mutual Hamming distance is 2. In some embodiments, the group of nucleic acid barcodes is designed such that the minimum mutual Hamming distance is 1.

[0050] In various embodiments, the present disclosure provides a method for determining in situ the presence, quantity, or localization of a target nucleic acid sequence (e.g., nucleic acid barcode) in a biological sample. In some embodiments, the methods being considered may include, in a biological sample, (a) reacting a DNA molecule containing the target nucleic acid sequence operably linked to a promoter configured to promote transcription of the target nucleic acid sequence with a sequence-specific RNA polymerase to produce an RNA molecule containing a sequence complementary to the target nucleic acid sequence; (b) reacting the RNA molecule with a reverse transcriptase to produce a cDNA molecule containing a copy of the target nucleic acid sequence; and (c) performing in situ sequencing (e.g., synthetic sequencing) to visualize the target nucleic acid sequence.

[0051] In various embodiments, the Disclosure provides a method for determining in situ the presence, quantity, and / or localization of a nucleic acid sequence of interest in one or more fixed mammalian cells in a biological sample, the method comprising (a) reverse transcribing a target RNA containing the nucleic acid sequence of interest in one or more fixed mammalian cells using DNA primers to generate a first cDNA molecule containing the nucleic acid sequence of interest, wherein the DNA primers comprise (i) a 5' nucleic acid sequence containing a sequence-specific RNA polymerase promoter, and (ii) a 3' nucleic acid sequence complementary to a portion of the target RNA adjacent to the nucleic acid sequence of interest. The method comprises: (b) generating a first cDNA molecule comprising a DNA primer hybridizing to a target RNA, the first cDNA molecule containing a sequence-specific RNA polymerase promoter operably linked to the nucleic acid sequence of interest; (c) reacting the first cDNA molecule with a sequence-specific RNA polymerase to generate an RNA transcript containing the nucleic acid sequence of interest; (d) reacting the RNA transcript with a reverse transcriptase to generate a second cDNA molecule containing the nucleic acid sequence of interest; and (d) sequencing the second cDNA molecule in situ to visualize the nucleic acid sequence of interest in one or more fixed mammalian cells.

[0052] Figure 1B shows an exemplary workflow of the method provided herein. Panel 100 illustrates a DNA molecule (101) containing a target nucleic acid sequence (102) (e.g., a nucleic acid barcode) operably linked to a promoter (103) configured to promote transcription of the target nucleic acid sequence in the presence of a sequence-specific RNA polymerase, within a fixed cell. Panel 110 shows the DNA molecule (101) reacting with a sequence-specific RNA polymerase (111) (e.g., T7 RNA polymerase) to generate an RNA molecule (112) containing a transcript of the target nucleic acid sequence (102). Panel 120 shows the RNA molecule (112) reacting with a reverse transcriptase (121) to generate a cDNA molecule (122) containing a copy of the target nucleic acid sequence. In panel 130, a cDNA molecule (122) undergoes synthetic sequencing and is reacted with sequencing polymerase (131) in the presence of detectably labeled nucleotides to produce a short amplicon containing detectably labeled nucleotides (132) that generates a detectable signal (133). The detectable signal (133) can be removed, allowing for further multiple rounds of detection of multiple target nucleic acid sequences.

[0053] In some embodiments, the DNA molecule is an exogenous DNA molecule. In some embodiments, the DNA molecule is an endogenous DNA molecule (e.g., genomic DNA or mitochondrial DNA). In some embodiments, the DNA molecule is derived from an exogenous nucleic acid sequence (e.g., introduced by viral transduction). In some embodiments, the DNA molecule is a cDNA molecule synthesized in situ from an exogenous DNA molecule. In some embodiments, the DNA molecule is a cDNA molecule synthesized in situ from an endogenous DNA molecule.

[0054] In various embodiments, the DNA molecule is produced by (i) a contact step of contacting an RNA molecule containing the nucleic acid sequence of the target with a DNA primer having a 5' end and a 3' end, wherein the DNA primer comprises A) a 3' nucleic acid sequence complementary to a portion of the nucleic acid molecule adjacent to the nucleic acid sequence of the target, and (B) a 5' nucleic acid sequence containing a promoter such that the DNA primer hybridizes to the nucleic acid sequence of the target; (ii) a step of performing reverse transcription to extend the DNA primer, thereby generating a single-stranded cDNA; and (iii) a step of converting the single-stranded cDNA to a double-stranded cDNA using double-strand synthesis, thereby generating a DNA molecule.

[0055] In a particular embodiment, the target nucleic acid sequence is contained within an RNA molecule (e.g., mRNA) within a cell in a biological sample. Figure 2C shows an exemplary workflow for introducing an exogenous promoter into an intracellular nucleic acid sequence. Panel 200 shows an RNA molecule (201) containing a transcript of the target nucleic acid sequence (202) within a fixed cell in a biological sample. Panel 210 shows a DNA primer (211) containing an exogenous sequence-specific promoter (103) ligated to a nucleic acid sequence (212) that hybridizes with a portion of the transcript of the target nucleic acid sequence (202). A reverse transcription reaction is performed to generate an ssDNA molecule (213) containing the target nucleic acid sequence (102) operably ligated to the exogenous sequence-specific promoter (103). The SSDNA molecule (213) can be used to generate dsDNA via an optional second-strand synthesis reaction. Furthermore, ssDNA molecules can also be synthesized using DNA primers (211) containing a double-stranded region with a sequence-specific promoter (103), thereby promoting RNA polymerase activity without second-strand synthesis. The ssDNA or dsDNA molecule can then be introduced into RNA synthesis, reverse transcription, and in-situ sequencing processes as shown in Figure 1B.

[0056] In some embodiments, the method includes contacting cells with RNase to degrade endogenous RNA molecules before reacting the DNA molecules with sequence-specific RNA polymerase.

[0057] Those skilled in the art will understand that, over a series of RNA synthesis or reverse transcriptions from a DNA template, the target nucleic acid sequence may exist at certain steps as the reverse complement of the original target nucleic acid sequence. For example, an RNA synthesis reaction using a DNA template containing the target nucleic acid sequence produces a product RNA molecule containing the reverse complement of the target nucleic acid sequence. The product RNA molecule is then reverse transcribed to produce a cDNA molecule containing the nucleic acid sequence, which is a copy of the original.

[0058] The disclosed method can detect the presence, quantity, or location of at least 100 target nucleic acid sequences, at least 90 target nucleic acid sequences, at least 80 target nucleic acid sequences, at least 70 target nucleic acid sequences, at least 60 target nucleic acid sequences, at least 50 target nucleic acid sequences, at least 40 target nucleic acid sequences, at least 30 target nucleic acid sequences, at least 20 target nucleic acid sequences, at least 15 target acid sequences, at least 10 target nucleic acid sequences, at least 9 target nucleic acid sequences, at least 8 target nucleic acid sequences, at least 7 target nucleic acid sequences, at least 6 target nucleic acid sequences, at least 5 target nucleic acid sequences, at least 4 target nucleic acid sequences, at least 3 target nucleic acid sequences, at least 2 target nucleic acid sequences, or at least 1 target nucleic acid sequence.

[0059] In some embodiments, multiple target nucleic acid sequences reside on a single nucleic acid molecule. In some embodiments, multiple nucleic acid sequences reside on multiple different nucleic acid molecules within a cell.

[0060] In certain embodiments, the sample is selected from tissue samples, liquid samples, and cell samples. In some embodiments, the biological sample is a tissue sample. In some embodiments, the biological sample is a liquid sample. In some embodiments, the sample is a cell sample. In some embodiments, the sample is a two-dimensional cell culture sample. In some embodiments, the sample is a three-dimensional cell culture sample. In some embodiments, the sample is a suspension cell culture sample. In some embodiments, the sample is an organoid sample. In some embodiments, the sample is a xenocellular culture sample. In some embodiments, the sample is a patient-derived cell sample. In some embodiments, the sample is a formalin-fixed paraffin-embedded (FFPE) tissue sample. In some embodiments, the sample is a cryopreserved tissue sample.

[0061] In certain embodiments, after removal of a detectable signal, RNA synthesis, reverse transcription, and in-situ sequencing are repeated one or more times, two or more times, three or more times, four or more times, five or more times, six or more times, seven or more times, eight or more times, nine or more times, ten or more times, fifteen or more times, twenty or more times, twenty-five or more times, thirty or more times, three or more times, forty or more times, forty or more times, or fifty or more times. In some embodiments, RNA synthesis, reverse transcription, and in-situ sequencing are repeated one or more times. In some embodiments, removal of a detectable label, re-probing, incorporation of the labeled nucleotide, and re-imaging are repeated two or more times. In some embodiments, RNA synthesis, reverse transcription, and in-situ sequencing are repeated three or more times. In some embodiments, RNA synthesis, reverse transcription, and in-situ sequencing are repeated four or more times. In some embodiments, RNA synthesis, reverse transcription, and in-situ sequencing are repeated five or more times. In some embodiments, RNA synthesis, reverse transcription, and in situ sequencing are repeated six or more times. In some embodiments, RNA synthesis, reverse transcription, and in situ sequencing are repeated seven or more times. In some embodiments, RNA synthesis, reverse transcription, and in situ sequencing are repeated eight or more times. In some embodiments, RNA synthesis, reverse transcription, and in situ sequencing are repeated nine or more times. In some embodiments, RNA synthesis, reverse transcription, and in situ sequencing are repeated ten or more times. In some embodiments, RNA synthesis, reverse transcription, and in situ sequencing are repeated fifteen or more times. In some embodiments, RNA synthesis, reverse transcription, and in situ sequencing are repeated twenty or more times. In some embodiments, RNA synthesis, reverse transcription, and in situ sequencing are repeated twenty-five or more times. In some embodiments, RNA synthesis, reverse transcription, and in situ sequencing are repeated thirty or more times. In some embodiments, RNA synthesis, reverse transcription, and in situ sequencing are repeated 35 or more times.In some embodiments, RNA synthesis, reverse transcription, and in situ sequencing are repeated 40 or more times. In some embodiments, RNA synthesis, reverse transcription, and in situ sequencing are repeated 45 or more times. In some embodiments, RNA synthesis, reverse transcription, and in situ sequencing are repeated 50 or more times.

[0062] Biological samples The systems and methods described herein can be used to detect the presence or absence of one or more target nucleic acid sequences in a biological sample, such as a cell sample or a tissue sample, or to quantify their amount.

[0063] The target nucleic acid sequence can be detected and / or quantified in various samples. In certain embodiments, the sample is derived from the subject.

[0064] The sample may be in any form that allows for the measurement of the target nucleic acid sequence. Therefore, the sample must be sufficient to process, such as by preparing thin sections, to enable the detection of the analyte. Accordingly, the sample may be fresh, preserved by appropriate cryogenic techniques, or preserved by non-cryogenic techniques.

[0065] In some embodiments, the sample is a bodily fluid sample, such as blood, serum, plasma, urine, saliva, cerebrospinal fluid, or interstitial fluid sample.

[0066] In various embodiments, the biological sample comprises one or more mammalian cells selected from stem cells, mesoderm cells, endoderm cells, ectoderm cells, cardiomyocytes, immune cells, epithelial cells, lung cells, Clara cells, Paneth cells, pancreatic cells, gastric cells, goblet cells, glandular cells, ductal cells, central atrial cells, brush border cells, endocrine cells, thyroid cells, pancreatic islet cells, mucinous cells, pituitary cells, neurons, sensory neurons, receptor neurons, neural progenitor cells, pyramidal cells, rod cells, interneurons, astrocytes, oligodendrocytes, ependymal cells, pituitary cells, adipocytes, lipid cells, kidney or urinary tract cells, germ cells, endothelial cells, extracellular matrix cells, contractile cells, skeletal muscle cells, cardiomyocytes, blood cells, germ cells, nurse cells, or stromal cells. In various embodiments, the biological sample comprises one or more immune cells. In some embodiments, the immune cells are T cells, NK cells, B cells, macrophages, dendritic cells, mast cells, monocytes, neutrophils, basophils, eosinophils, hematopoietic stem cells, or immortalized immune cells. In some embodiments, the immune cells are T cells, NK cells, or B cells, mast cells, dendritic cells, macrophages, neutrophils, basophils, and / or eosinophils. In some embodiments, the immune cells express one or more cell therapy constructs (e.g., engineered immune receptors). In some embodiments, the immune cells encode chimeric antigen receptors (CARs). In some embodiments, the immune cells are chimeric antigen receptor-expressing T (CAR-T) cells or chimeric antigen receptor-expressing NK (CAR-NK) cells. In some embodiments, the immune cells are chimeric antigen receptor-expressing T (CAR-T) cells. In some embodiments, the immune cells are chimeric antigen receptor-expressing NK (CAR-NK) cells. In some embodiments, the biological sample includes embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), or cells derived from ESCs or iPSCs that have been selectively differentiated into a specific lineage.

[0067] In some embodiments, the sample is a tissue sample, such as a biopsy sample. Biopsy samples can be obtained using conventional biopsy instruments and procedures. Endoscopic biopsy, excision biopsy, incision biopsy, fine-needle biopsy, punch biopsy, shave biopsy, and skin biopsy are examples of recognized medical procedures that can be used by those skilled in the art to obtain tissue samples. The standard process for handling clinical biopsy tissue specimens is to fix the tissue sample in formalin and then embed the sample in paraffin. This form of sample is commonly known as formalin-fixed paraffin-embedded (FFPE) tissue. Appropriate techniques for tissue preparation for subsequent analysis are well known to those skilled in the art.

[0068] In certain embodiments, the sample is a cell sample or a cell supernatant sample.

[0069] In some embodiments, the biological sample comprises one or more cells. In some embodiments, the biological sample comprises one or more cells composed of cells derived from a single species. In some embodiments, the biological sample comprises one or more cells derived from multiple species. In some embodiments, the biological sample comprises human cells and mouse cells. In some embodiments, the biological sample comprises human immune cells and mouse cells. In some embodiments, the biological sample comprises one or more cancer cells or fibroblasts. In some embodiments, the biological sample comprises one or more human cancer cells. In some embodiments, the biological sample comprises one or more mouse cancer cells.

[0070] In various embodiments, the biological sample is immobilized. In some embodiments, the biological sample is immobilized using a solution containing formaldehyde and / or paraformaldehyde. In some embodiments, the biological sample is immobilized using a solution containing more than 1% paraformaldehyde (w / v). In some embodiments, the biological sample is immobilized using a sample containing 4% paraformaldehyde. In some embodiments, the sample is an FFPE tissue sample.

[0071] In some embodiments, the biological sample is fixed by cryopreservation (e.g., using liquid nitrogen). In some embodiments, the cryopreserved biological sample comprises an optimal cutting temperature (OCT) compound, a hydrogel matrix, or a swellable polymer hydrogel. In some embodiments, the cryopreserved sample comprises an optimal cutting temperature (OCT) compound. In some embodiments, the sample comprises a hydrogel matrix. In some embodiments, the sample comprises a swellable polymer hydrogel.

[0072] In some embodiments, the sample is fixed using a solution containing alcohol. In some embodiments, the alcohol is methanol or ethanol. In some embodiments, the alcohol is methanol. In some embodiments, the alcohol is ethanol. In some embodiments, the sample is fixed using a solution containing glutaraldehyde.

[0073] nucleic acid sequence In certain embodiments, the methods disclosed herein relate to determining the presence, quantity, and / or localization of one or more nucleic acid sequences of interest. Examples of nucleic acids include DNA, mRNA, premRNA, nascent RNA, transfer RNA, antisense oligonucleotides, siRNA, miRNA, tmRNA, snRNA, piRNA, sRNA, circular RNA, snoRNA, eRNA, pRNA, sgRNA, gRNA (e.g., CRISPR-mediated gene editing applications or CRISPR-mediated gene activation or silencing applications), and fabricated DNA or RNA (e.g., for therapeutic, screening, or basic scientific purposes). In some embodiments, the nucleic acid sequence of interest is located on a complementary DNA (cDNA) molecule produced by reverse transcribing an RNA molecule containing the nucleic acid sequence of interest.

[0074] In some embodiments, the nucleic acid sequence of interest is a nucleic acid barcode. A “nucleic acid barcode” or “barcode” (also called a “unique molecular identifier” or “UMI”) may refer to a random or predetermined nucleotide sequence of a specified length that can be used to identify a particular cell or cell type. Nucleic acid barcodes are useful in many applications, including sequencing techniques. Further details regarding nucleic acid barcodes can be found, for example, in U.S. Patent No. 8,053,192, PCT Publication No. WO2005 / 068656, and U.S. Patent Publication No. 2013 / 0274117.

[0075] In various embodiments, the nucleic acid sequence of interest is located on an exogenous nucleic acid molecule or on a nucleic acid molecule derived from an exogenous nucleic acid sequence. In some embodiments, the exogenous nucleic acid is introduced into one or more mammalian cells before fixation. The exogenous nucleic acid can be introduced into cells by any suitable method known in the art. In some embodiments, the exogenous nucleic acid is introduced into one or more mammalian cells by viral transduction, site-specific nuclease, or site-specific recombinase. In some embodiments, the exogenous nucleic acid is introduced into one or more mammalian cells by viral transduction. In some embodiments, the exogenous nucleic acid is introduced into one or more mammalian cells by site-specific nuclease. In some embodiments, the exogenous nucleic acid is introduced into one or more mammalian cells by site-specific recombinase. In some embodiments, the exogenous nucleic acid sequence is integrated into the genome of one or more mammalian cells. In some embodiments, the exogenous nucleic acid sequence is integrated into a specific site (i.e., a pre-specified site) within the genome of one or more mammalian cells. In some embodiments, the exogenous nucleic acid is integrated into a random site within the genome of one or more mammalian cells. In some embodiments, exogenous nucleic acids are incorporated into one or more mammalian cells using a viral vector. In some embodiments, the viral vector is selected from lentiviral vectors, retroviral vectors, adenovirus vectors, HSV vectors, baculovirus vectors, virus-like particles, pseudotyped virus-like capsids, oncolytic viral vectors, or adeno-associated virus (AAV) vectors. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is an adenovirus vector. In some embodiments, the viral vector is an HSV vector. In some embodiments, the viral vector is a baculovirus vector. In some embodiments, the viral vector is a virus-like particle. In some embodiments, the viral vector is an HSV vector.In some embodiments, the viral vector is an oncolytic viral vector. In some embodiments, the viral vector is a pseudotyped virus-like capsid. In some embodiments, the viral vector is an AAV vector.

[0076] In some embodiments, the exogenous nucleic acid is not integrated into the chromosome of one or more mammalian cells. In some embodiments, the exogenous nucleic acid is not integrated into the chromosome of one or more mammalian cells but is retained within the nucleus of one or more mammalian cells. In some embodiments, the exogenous nucleic acid molecule is contained within a plasmid or artificial chromosome.

[0077] In various embodiments, the nucleic acid sequence of interest is an endogenous nucleic acid sequence. In some embodiments, the nucleic acid sequence of interest is an endogenous nucleic acid sequence operably linked to an exogenous promoter. In some embodiments, the nucleic acid sequence of interest is an endogenous nucleic acid sequence that changes between cells in a biological sample. In some embodiments, the nucleic acid sequence of interest is an endogenous nucleic acid sequence that does not change between cells in a biological sample. Where provided herein, the nucleic acid sequence of interest may also include sequences other than the sequence of interest.

[0078] In some embodiments, the target nucleic acid sequence is less than 100 nucleotides, less than 90 nucleotides, less than 80 nucleotides, less than 70 nucleotides, less than 60 nucleotides, less than 50 nucleotides, less than 40 nucleotides, less than 30 nucleotides, less than 25 nucleotides, less than 20 nucleotides, less than 15 nucleotides, less than 10 nucleotides, or less than 5 nucleotides. In some embodiments, the target nucleic acid sequence is less than 100 nucleotides. In some embodiments, the target nucleic acid sequence is less than 90 nucleotides. In some embodiments, the target nucleic acid sequence is less than 80 nucleotides. In some embodiments, the target nucleic acid sequence is less than 70 nucleotides. In some embodiments, the target nucleic acid sequence is less than 60 nucleotides. In some embodiments, the target nucleic acid sequence is less than 50 nucleotides. In some embodiments, the target nucleic acid sequence is less than 40 nucleotides. In some embodiments, the target nucleic acid sequence is less than 30 nucleotides. In some embodiments, the target nucleic acid sequence is less than 25 nucleotides. In some embodiments, the target nucleic acid sequence is less than 20 nucleotides long. In some embodiments, the target nucleic acid sequence is less than 15 nucleotides long. In some embodiments, the target nucleic acid sequence is less than 10 nucleotides long. In some embodiments, the target nucleic acid sequence is less than 5 nucleotides long.

[0079] In some embodiments, the nucleic acid sequence is present in all or substantially all cells in the biological sample. In some embodiments, the nucleic acid sequence of interest is present in less than 100% of the cells in the biological sample. In some embodiments, the nucleic acid sequence of interest is present in less than 90% of the cells in the biological sample. In some embodiments, the nucleic acid sequence of interest is present in less than 80% of the cells in the biological sample. In some embodiments, the nucleic acid sequence of interest is present in less than 70% of the cells in the biological sample. In some embodiments, the nucleic acid sequence of interest is present in less than 60% of the cells in the biological sample. In some embodiments, the nucleic acid sequence of interest is present in less than 50% of the cells in the biological sample. In some embodiments, the nucleic acid sequence of interest is present in less than 40% of the cells in the biological sample. In some embodiments, the nucleic acid sequence of interest is present in less than 30% of the cells in the biological sample. In some embodiments, the nucleic acid sequence of interest is present in less than 20% of the cells in the biological sample. In some embodiments, the nucleic acid sequence of interest is present in less than 10% of the cells in the biological sample.

[0080] In some embodiments, the nucleic acid sequence of interest is contained within a nucleic acid molecule further comprising nucleic acid sequences encoding one or more proteins. In some embodiments, the DNA molecule further comprises one or more polynucleotide sequences encoding an exogenous protein, an endogenous protein, or a mixture of exogenous and endogenous proteins. In some embodiments, the one or more proteins comprise one or more exogenous proteins. In some embodiments, the expression of one or more exogenous proteins is controlled by one or more endogenous proteins in the mammalian cell.

[0081] In some embodiments, one or more exogenous proteins are synthetic proteins and / or chimeric proteins. In some embodiments, one or more exogenous proteins are independently selected from the group consisting of chimeric antigen receptors (CARs), antibodies, T cell receptors, cytokines, cell surface receptors, transcription factors, signaling proteins, and proteases. In some embodiments, one or more exogenous proteins include CARs. In some embodiments, one or more exogenous proteins include antibodies. In some embodiments, one or more exogenous proteins include antibodies. In some embodiments, one or more exogenous proteins include T cell receptors. In some embodiments, one or more exogenous proteins include cytokines. In some embodiments, one or more exogenous proteins include cell surface receptors. In some embodiments, one or more exogenous proteins include transcription factors. In some embodiments, one or more exogenous proteins include signaling proteins. In some embodiments, one or more exogenous proteins include proteases.

[0082] In some embodiments, two or more exogenous proteins are expressed. In some embodiments, three or more exogenous proteins are expressed. In some embodiments, four or more exogenous proteins are expressed. In some embodiments, five or more exogenous proteins are expressed. In some embodiments, six or more exogenous proteins are expressed. In some embodiments, seven or more exogenous proteins are expressed. In some embodiments, eight or more exogenous proteins are expressed. In some embodiments, nine or more exogenous proteins are expressed. In some embodiments, ten or more exogenous proteins are expressed.

[0083] In some embodiments, the nucleic acid sequence of interest is contained within a nucleic acid molecule further comprising a nucleic acid sequence encoding an endogenous protein. In some embodiments, the nucleic acid sequence of interest is contained within a nucleic acid molecule further comprising a nucleic acid sequence encoding an exogenous RNA. In some embodiments, the nucleic acid sequence of interest is contained within a nucleic acid molecule further comprising a nucleic acid sequence encoding an endogenous RNA.

[0084] In some embodiments, the nucleic acid sequence of interest is contained within a nucleic acid molecule further comprising a nucleic acid sequence encoding a viral genome. In some embodiments, the viral genome is an oncolytic viral genome. In some embodiments, the oncolytic viral genome is the genome of an adenovirus, herpes simplex virus (HSV), parvovirus, or poxvirus (e.g., vaccinia virus or myxoma virus).

[0085] In some embodiments, the DNA molecule further comprises a polynucleotide sequence encoding a nucleic acid sequence that modifies the expression, function, and / or sequence of one or more genes. In some embodiments, the DNA molecule further comprises a polynucleotide sequence encoding a nucleic acid sequence that contributes to modifying the genome of a cell. In some embodiments, modifying the genome of a cell comprises introducing one or more mutations into the genome of a cell. In some embodiments, one or more mutations include one or more nucleic acid insertions, nucleic acid deletions, frameshift mutations, missense mutations, and nonsense mutations. In some embodiments, the target RNA encodes a sequence that can modify the expression of one or more genes, either alone or as a component of a gene editing system (e.g., CRISPR). In some embodiments, the nucleic acid sequence that modifies the expression, function, and / or sequence of one or more genes is selected from the group consisting of sgRNA (e.g., in a CRISPR construct), gRNA, shRNA, and miRNA. In some embodiments, the nucleic acid sequence that modifies the expression of one or more genes is sgRNA. In some embodiments, the nucleic acid sequence that modifies the expression, function, and / or sequence of one or more genes is gRNA. In some embodiments, the nucleic acid sequence that modifies the expression, function, and / or sequence of one or more genes is shRNA. In some embodiments, the nucleic acid sequence that alters the expression, function, and / or sequence of one or more genes is a miRNA.

[0086] Promoter and sequence-specific RNA polymerase In various embodiments, the methods disclosed herein involve reacting a DNA molecule containing a sequence-specific RNA polymerase promoter operably linked to a nucleic acid sequence of interest with a sequence-specific RNA polymerase to drive transcription of the nucleic acid sequence of interest in one or more mammalian cells in a biological sample. Using the sequence-specific RNA polymerase and promoter, one or more target nucleic acid sequences of interest can be selectively amplified with minimal or substantially no off-target RNA synthesis. In some embodiments, the methods disclosed herein include reacting the biological sample with an RNase (e.g., degrading endogenous RNA) before adding the sequence-specific RNA polymerase.

[0087] In some embodiments, one or more mammalian cells contain a single target nucleic acid sequence operably ligated to a sequence-specific RNA polymerase promoter configured to drive the transcription of the target nucleic acid sequence in the presence of a sequence-specific RNA polymerase. In some embodiments, one or more mammalian cells contain multiple target nucleic acid sequences, each operably ligated to a sequence-specific RNA polymerase promoter configured to drive the transcription of each target nucleic acid sequence in the presence of a sequence-specific RNA polymerase. In some embodiments, each sequence-specific RNA polymerase and promoter is identical. In some embodiments, each sequence-specific RNA polymerase and promoter is different. In some embodiments, some sequence-specific RNA polymerases and promoters are identical, and some are different.

[0088] In some embodiments, one or more mammalian cells include a first nucleic acid sequence of interest operably ligated to a first sequence-specific RNA polymerase promoter configured to drive the transcription of the first nucleic acid sequence of interest in the presence of a first sequence-specific RNA, and a second nucleic acid sequence of interest operably ligated to a second sequence-specific RNA polymerase promoter configured to drive the transcription of the nucleic acid sequence of interest in the presence of a second sequence-specific RNA polymerase. In some embodiments, the first and second promoters and RNA polymerases are the same. In some embodiments, the first and second promoters and RNA polymerases are different.

[0089] In some embodiments, one or more mammalian cells include three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more sequence-specific RNA polymerase promoters, each configured to drive the transcription of a distinct nucleic acid sequence of interest in the presence of a distinct sequence-specific RNA polymerase. In some embodiments, one or more mammalian cells include three or more sequence-specific RNA polymerase promoters, each configured to drive the transcription of a distinct nucleic acid sequence of interest in the presence of a distinct sequence-specific RNA polymerase. In some embodiments, one or more mammalian cells include four or more sequence-specific RNA polymerase promoters, each configured to drive the transcription of a distinct nucleic acid sequence of interest in the presence of a distinct sequence-specific RNA polymerase. In some embodiments, one or more mammalian cells include five or more sequence-specific RNA polymerase promoters, each configured to drive the transcription of a distinct nucleic acid sequence of interest in the presence of a distinct sequence-specific RNA polymerase. In some embodiments, one or more sequence-specific RNA polymerase promoters include six or more sequence-specific RNA polymerase promoters, each configured to drive the transcription of a distinct nucleic acid sequence of interest in the presence of a distinct sequence-specific RNA polymerase. In some embodiments, the system includes seven or more sequence-specific RNA polymerase promoters, each configured to drive the transcription of a distinct nucleic acid sequence of interest in the presence of a distinct sequence-specific RNA polymerase. In some embodiments, the system includes eight or more sequence-specific RNA polymerase promoters, each configured to drive the transcription of a distinct nucleic acid sequence of interest in the presence of a distinct sequence-specific RNA polymerase. In some embodiments, the system includes nine or more sequence-specific RNA polymerase promoters, each configured to drive the transcription of a distinct nucleic acid sequence of interest in the presence of a distinct sequence-specific RNA polymerase. In some embodiments, the system includes ten or more sequence-specific RNA polymerase promoters, each configured to drive the transcription of a distinct nucleic acid sequence of interest in the presence of a distinct sequence-specific RNA polymerase.

[0090] In some embodiments, the RNA polymerase is a DNA-dependent RNA polymerase. In some embodiments, the sequence-specific RNA polymerase promoter is a transcriptionally active variant of a known sequence-specific RNA polymerase promoter (i.e., a promoter containing a mutant sequence relative to a known sequence but possessing transcriptional activity). In some embodiments, the sequence-specific RNA polymerase promoter is a phage promoter or a transcriptionally active variant thereof, and the sequence-specific RNA polymerase is a phage RNA polymerase (i.e., a bacteriophage-derived promoter). In some embodiments, the sequence-specific RNA polymerase promoter and the sequence-specific RNA polymerase are selected from the group consisting of the T7 promoter or a transcriptionally active variant thereof and the T7 RNA polymerase; the T3 promoter or a transcriptionally active variant thereof and the T3 RNA polymerase; and the SP6 promoter or a transcriptionally active variant thereof and the SP6 RNA polymerase. In some embodiments, the promoter is the T7 promoter or a transcriptionally active variant thereof, and the RNA polymerase is the T7 RNA polymerase. In some embodiments, the promoter is the T3 promoter or a transcriptionally active variant thereof, and the RNA polymerase is the T3 RNA polymerase. In some embodiments, the promoter is the SP6 promoter or a transcriptionally active variant thereof, and the RNA polymerase is the SP6 RNA polymerase.

[0091] In some embodiments, the sequence-specific RNA polymerase promoter is a bacterial promoter or a transcriptional variant thereof, and the sequence-specific RNA polymerase is a bacterial RNA polymerase. In some embodiments, the sequence-specific RNA polymerase promoter is a eukaryotic promoter or a transcriptional variant thereof, and the sequence-specific RNA polymerase is a eukaryotic RNA polymerase. In some embodiments, the sequence-specific RNA polymerase promoter is a viral promoter or a transcriptional variant thereof, and the sequence-specific RNA polymerase is a viral RNA polymerase. In some embodiments, the sequence-specific RNA polymerase promoter is a synthetic promoter, and the sequence-specific RNA polymerase is a synthetic RNA polymerase.

[0092] In some embodiments, the sequence-specific promoter is contained within an exogenous nucleic acid molecule or a nucleic acid molecule derived from an exogenous nucleic acid molecule. In some embodiments, the exogenous nucleic acid molecule further comprises a transcription terminator. In some embodiments, the transcription terminator is a T7 transcription terminator, a T3 transcription terminator, or an SP6 transcription terminator. In some embodiments, the transcription terminator is a T7 transcription terminator. In some embodiments, the transcription terminator is a T3 transcription terminator. In some embodiments, the transcription terminator is an SP6 transcription terminator.

[0093] In some embodiments, the sequence-specific promoter is an exogenous promoter (e.g., the T7 promoter) introduced into cells to control the expression of an endogenous nucleic acid sequence of interest. In some embodiments, the exogenous promoter is introduced by (1) introducing a DNA primer into one or more mammalian cells that includes (a) a 5' nucleic acid sequence containing the exogenous promoter and (b) a 3' nucleic acid sequence complementary to a portion of the target RNA containing the nucleic acid sequence of interest, and (2) performing a reverse transcription reaction to synthesize a cDNA molecule containing the exogenous primer operably linked to the nucleic acid sequence of interest. In some embodiments, a second strand synthesis reaction is performed to convert the cDNA molecule to double-stranded DNA (dsDNA) (Figure 2A). In some embodiments, no second strand synthesis reaction is performed, and the DNA primer includes a dsDNA portion containing the exogenous promoter so that the reverse transcription reaction of the cDNA molecule can be directly driven using the cDNA (Figure 2B). In some embodiments, the dsDNA portion of the DNA primer is hybridized dsDNA or a hairpin. In some embodiments, the dsDNA portion of the DNA primer is hybridized dsDNA. In some embodiments, the dsDNA portion of the DNA primer is a hairpin.

[0094] In some embodiments, the exogenous promoter is integrated into the cell's genome. In some embodiments, the exogenous promoter is integrated into a specific site within the cell's genome. In some embodiments, the exogenous promoter is integrated into a random site within the genome of one or more mammalian cells. In some embodiments, the exogenous promoter is integrated into the cell's genome by a site-specific nuclease. In some embodiments, the exogenous promoter is integrated into the cell's genome by a site-specific recombinant enzyme.

[0095] In some embodiments, multiple different exogenous promoters are integrated into the cell's genome. In some embodiments, each exogenous promoter is integrated into a specific site within the cell's genome. In some embodiments, some exogenous promoters are integrated into specific sites within the cell's genome, while others are integrated into random sites within the cell's genome.

[0096] In some embodiments, the exogenous promoter is a sequence-specific RNA polymerase promoter. In some embodiments, the exogenous promoter is a phage promoter. In some embodiments, the exogenous promoter is selected from the group consisting of the T7 promoter, the T3 promoter, and the SP6 promoter. In some embodiments, the exogenous promoter is the T7 promoter. In some embodiments, the exogenous promoter is the T3 promoter. In some embodiments, the exogenous promoter is the SP6 promoter.

[0097] Reverse transcription In some embodiments, the methods disclosed herein involve performing a reverse transcription reaction to produce a cDNA molecule of a transcript produced by a sequence-specific RNA polymerase. Reverse transcription generally involves reacting an RNA molecule and a DNA primer with a reverse transcriptase to produce a cDNA molecule. Reverse transcription can be carried out using any suitable method known in the art. In some embodiments, the DNA primer is a random primer. In some embodiments, the DNA primer is a sequence-specific primer. In some embodiments, the reverse transcriptase is AMV reverse transcriptase. In some embodiments, the reverse transcriptase is MMLV reverse transcriptase. In some embodiments, the reverse transcriptase is a manipulated reverse transcriptase.

[0098] In situ sequencing In various embodiments, the methods disclosed herein include detection of a target nucleic acid by one or more in-situ sequencing techniques (e.g., sequencing by synthesis, sequencing by ligation, or sequencing by avidity). In-situ sequencing techniques generally involve incorporating a nucleotide or oligonucleotide containing a detectable label (e.g., a fluorescent dye containing a fluorophore) into a nucleic acid complementary to a template nucleic acid (e.g., a nucleic acid sequence of interest).

[0099] In some embodiments, nucleic acid sequences can be detected via synthetic sequencing. Synthetic sequencing can be performed using an enzyme having DNA polymerase activity. Synthetic sequencing is typically performed using an enzyme having DNA polymerase activity to incorporate one or more labeled nucleotides, where the labeled nucleotides include a detectable label (e.g., a fluorescent label) and optionally a cleavable strand terminator modification. In some embodiments, the detectable label is a cleavable detectable label. In each round, a mixed population of multiple nucleotides, or a population containing a single nucleotide type, is incorporated onto the free 3' end of the target nucleic acid to be sequenced. Imaging is performed to identify the detectable label added to the target nucleic acid, label removal is performed (e.g., cleavage, photobleaching, dissociation by changing buffer conditions, etc.), and nucleotides from another round are added to the target nucleic acid.

[0100] In some embodiments, nucleotides containing chain terminator modifications are also incorporated into the probe to prevent the incorporation of additional nucleotides. Preventing the incorporation of additional nucleotides into the first set of probes may be useful in enabling the labeling and detection of additional analytes in successive rounds by preventing the incorporation of labeled nucleotides into previously detected probes. In some embodiments, the chain terminator is irreversible. In some embodiments, the chain terminator is reversible. In some embodiments, the chain terminator is present in nucleotides containing a detectable label. In some embodiments, the chain terminator is present in additional nucleotides that do not contain a detectable label.

[0101] In some embodiments, ligation sequencing can be performed using oligonucleotides containing degenerate bases (e.g., via an oligonucleotide pool) and incorporated using a ligation reaction. Ligation sequencing can be performed using labeled oligonucleotide species (often a pool, e.g., degenerate bases excluding the site(s) to be sequenced), and unlike synthetic sequencing, the use of an enzyme with DNA ligase activity facilitates incorporation into the analyte. Both cleavable, detectable labels and reversible strand terminators can still be used, as in synthetic sequencing. To sequence to degenerate bases within the oligonucleotides, the anchor sequences and ligated oligonucleotides can be removed from the analyte, and a new set can be incorporated into subsequent rounds.

[0102] In some embodiments, avidity sequencing can be performed by binding a detectably labeled polymer-nucleotide substrate ("avidite"), each containing multiple copies of a single nucleotide, to a DNA template. A manipulated DNA polymerase binds to the template DNA and facilitates the specific binding of the avidite to congener nucleotides without incorporating the avidite into the template DNA or synthesizing a complementary strand. The detectably labeled avidite can then be visualized and subsequently removed by washing, allowing for a subsequent detection round. In some embodiments, the detectable label on the avidite is a cleavable detectable label. Further details of avidity sequencing can be found in Arslan, S. et al. Sequencing by avidity enables high accuracy with low reagent consumption. Nat Biotechnol (2023).

[0103] In some embodiments, the methods disclosed herein include a rolling circle amplification reaction performed on a cDNA molecule containing the nucleic acid sequence of interest prior to in situ sequencing. Information regarding rolling circle amplification is provided in Schweitzer, et al. PNAS. 2000 Aug 29;97(18):10113-9.

[0104] In some embodiments, the nucleic acid sequence of interest is flanked by a first padlock binding sequence and a second padlock binding sequence. In some embodiments, the method disclosed herein includes: (1) contacting cDNA with a first padlock probe having a 5' end and a 3' end, wherein the first padlock probe includes a 5' nucleic acid sequence inversely complementary to the first padlock binding site and a 3' nucleic acid sequence inversely complementary to the second padlock binding site, so that the 5' and 3' nucleic acid sequences can hybridize to the cDNA; (2) extending the 3' end of the first padlock probe via the nucleic acid sequence of interest using DNA polymerase; (3) generating a circular DNA template containing the nucleic acid sequence inversely complementary to the nucleic acid sequence of interest by ligating the 5' end of the padlock probe to the extended 3' end of the padlock probe; and (4) generating additional copies of the nucleic acid sequence of interest using rolling circle amplification of the DNA template.

[0105] In some embodiments, multiple target nucleic acid sequences are each flanked by padlock binding sites. In some embodiments, the pair of padlock binding sites flanking each target nucleic acid sequence is identical. In some embodiments, the pair of padlock binding sites flanking each target nucleic acid sequence is different. In some embodiments, the first target nucleic acid sequence is flanked by a first padlock binding site and a second padlock binding site, and the second target nucleic acid sequence is flanked by a third padlock binding site and a fourth padlock binding site. In some embodiments, the first and second padlock binding sites are identical to the third and fourth padlock binding sites. In some embodiments, the first and second padlock binding sites are different from the third and fourth padlock binding sites. In some embodiments, the methods disclosed herein include (1) contacting cDNA with a second padlock probe having a 5' end and a 3' end, wherein the second padlock probe has a 5' nucleic acid sequence inversely complementary to a third padlock binding site and a 3' nucleic acid sequence inversely complementary to a fourth padlock binding site; (2) extending the 3' end of the second padlock probe through the second target nucleic acid sequence using DNA polymerase; (3) generating a circular DNA template containing the nucleic acid sequence inversely complementary to the second target nucleic acid sequence by ligating the 5' end of the second padlock probe to the extended 3' end of the second padlock probe; and (4) generating additional copies of the second target nucleic acid sequence using rolling circle amplification of the DNA template.

[0106] Detectable signs In some embodiments, the detectable labels used in the present invention include fluorescent dyes. Fluorescent dyes are widely used in biological research and medical diagnostics. In particular, the diversity of fluorophores with distinguishable color ranges has made it more practical to perform multiplex assays that can detect multiple biological targets simultaneously. The ability to visualize multiple targets in parallel is often required to depict the spatial and temporal relationships between different biological targets in vitro and in vivo.

[0107] In some embodiments, the fluorescent dye is Alexa Fluor. Examples of Alexa Fluor include, but are not limited to, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, and Alexa Fluor 790.

[0108] In some embodiments, the fluorescent dye is a rhodamine dye. Examples of rhodamine dyes include, but are not limited to, rhodamine, rhodamine 6G, rhodamine 123, rhodamine B, sulforhodamine 101, and sulforhodamine B.

[0109] In some embodiments, the fluorescent dye is DyLight Electro. Examples of DyLight Electro include, but are not limited to, DyLight 350, DyLight 405, DyLight 488, DyLight 550, DyLight 594, DyLight 633, DyLight 650, DyLight 680, DyLight 755, and DyLight 800.

[0110] In some embodiments, the fluorescent dye is a cyanine dye. Examples of cyanine dyes include, but are not limited to, cyanine 2 (Cy2), cyanine 3 (Cy3), cyanine 3B (Cy3B), cyanine 3.5 (Cy3.5), cyanine 5 (Cy5), cyanine 5.5 (Cy5.5), cyanine 7 (Cy7), and cyanine 7.5 (Cy7.5).

[0111] In some embodiments, the fluorescent dye is an ATTO dye. Examples of ATTO dyes are ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 540Q, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO 580Q, ATTO Rho101, ATTO 590, ATTO Rho13, ATTO 594, ATTO 610, ATTO 612Q, ATTO 620, ATTO Rho14, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700, ATTO Examples include, but are not limited to, the 725, ATTO 740, and ATTO MB2.

[0112] Other examples of fluorescent dyes include, but are not limited to, Freedom Dyes, Janelia Fluor Dyes, green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), DSRed, eGFP, mMerald, mWasabi, Azami Green, mAzurite, mCerulean, mTurquoise, mTopaz, mVenus, mCitrine, mBanana, Kusabia Orange, mOrange, dTomato, mTangerine, mRuby, mApple, mStrawberry, mCherry, mRaspberry, mPlum, fluorescein, phycoerythrin (PE), and peridinine chlorophyll protein (PerCP).

[0113] The fluorescent dye can be detected by any suitable method known to those skilled in the art, preferably by fluorescence microscopy. In some embodiments, the fluorescence microscopy is wide-field fluorescence microscopy. In some embodiments, the fluorescence microscopy is laser scanning confocal microscopy. In some embodiments, the fluorescence microscopy is spinning disk confocal microscopy. In some embodiments, the fluorescence microscopy is two-photon microscopy. The methods of fluorescence microscopy are summarized in Sanderson et al., Cold Spring Harb Protoc. 2014 Oct;2014(10).

[0114] In some embodiments, the detectable label of the present invention can include a radioisotope. The detectable label can be incorporated directly or indirectly by incorporating the label via a chelating agent, where the chelating agent is incorporated into the compound. Further, the label can be included as an additional substituent (group, moiety, position) to the compounds of the present invention or as an alternative substituent to any existing substituent. Exemplary radioisotopes include 3 H, 11 C, 14 C, 18 F, 32 P, 35 S, 123 I, 125 I, 131 I, 124 I, 19 F, 75 Br, 13 C, 13 N, 15 O, 76 Br, or 99 Tc. The radiolabel can occur at any substituent (group, moiety, position) on the compounds or probes of the present invention.

[0115] In various embodiments, the detectable label may be cleaved from a labeled nucleotide oligonucleotide or an avidite (i.e., a cleavable detectable label). In some embodiments, the detectable label is cleavable between a dye and a nitrogenous base. In some embodiments, the detectable label is cleavable between a dye and a sugar phosphate backbone. In some embodiments, the cleavable detectable label is cleavable between an oligonucleotide and a labeled nucleotide. In some embodiments, the cleavable detectable label produces the same or similar detectable signal. In some embodiments, the cleavable detectable label is a fluorophore having substantially overlapping excitation and / or emission spectra.

[0116] In some embodiments, the cleavable detectable label is cleaved by exposure to light. In some embodiments, the cleavable detectable label is cleaved by treatment with an acidic solution. In some embodiments, the cleavable detectable label is cleaved by treatment with an alkaline solution. In some embodiments, the cleavable detectable label is cleaved by treatment with a reducing agent. In some embodiments, the reducing agent is DTT or TCEP. In some embodiments, the cleavable detectable label is cleaved by treatment with a nuclease or DNA repair enzyme. In some embodiments, the cleavable detectable label is cleaved by treatment with palladium.

[0117] In some embodiments, the signal from the detectable label can be removed by incubation with a quencher. Examples of quenchers include, but are not limited to, Iowa Black, Dark Quencher, Black Hole Quencher, ZEN Quencher, Dabcyl, BHQ Quencher, BBQ Quencher, Atto Quencher, TAMRA, and MGB.

[0118] Throughout this description, where a composition is described as having, encompassing, or including a particular component, or where a process and method is described as having, encompassing, or including a particular step, it is also intended that there exist compositions of the present invention that are essentially composed of or consist of the listed components, and processes and methods of the present invention that are essentially composed of or consist of the listed processing steps.

[0119] Image processing In various embodiments, images are processed before analysis. Image processing can be performed using any suitable method known in the art. Further details regarding image processing for fluorescence microscopy are provided in Sanderson et al., Cold Spring Harb Protoc. 2014 Oct;2014(10), Baggett DW, et al. Front Bioinform. 2022 Jun 6;2:897238, Hallou A, et al. Development. 2021 Sep 15;148(18):dev199616, and Uchida S. Dev Growth Differ. 2013 May;55(4):523-49. In some embodiments, images are processed to reduce or remove background noise. In some embodiments, background noise is reduced or removed by applying a Laplacian of Gaussian filter. In some embodiments, different Laplacian of Gaussian filters are applied to each image to achieve a nearly uniform background noise level between images. In some embodiments, further filters are applied to remove anomalous or artificial signals. In some embodiments, the anomalous or artificial signals are small spots and / or dim spots. In some embodiments, the anomalous or artificial signals are identified to be removed by matching spots that are present at similar intensity across multiple channels.

[0120] In some embodiments, signals present in the image after background subtraction (e.g., one or more spots) are mapped to cells. In some embodiments, the mapped signals are reconstructed using a cell identification and joining algorithm. In some embodiments, the joining algorithm is a fuzzy join.

[0121] In some embodiments, loci representing T7 amplification signals are detected manually. In some embodiments, loci representing T7 amplification signals are detected by applying a minimum size threshold and / or colocalization to the nucleus. In some embodiments, loci representing T7 amplification signals are detected by deep learning using manually labeled training data. Further details of deep learning applications for image classification are provided in Pachitariu, M. & Stringer, C., Nat Methods 19, 1634-1641 (2022).

[0122] Detection of additional analytes In various embodiments, the methods provided herein further include the detection of additional analytes (i.e., analytes other than the nucleic acid sequence of interest). In some embodiments, the additional analytes include one or more of the following: proteins, RNA, DNA stained in a non-sequence-specific manner, DNA having a specific sequence, DNA mutations, lipids including but not limited to phospholipids and sphingolipids, carbohydrates including but not limited to monosaccharides and polysaccharides, metabolites, small molecules, cellular structures, and tissue structures. The additional analytes can be detected by any preferred method known in the art.

[0123] In some embodiments, the method further includes detecting the presence, quantity, and / or localization of one or more protein analytes within a cell. In some embodiments, one or more protein analytes are detected by immunofluorescence microscopy, direct immunofluorescence microscopy, indirect immunofluorescence microscopy, fluorescence-detectable DNA conjugate antibodies (e.g., CODEX system or Immuno-SABER), hybridization chain reaction (HCR) immunofluorescence microscopy, mass cytometry, aptamer-based protein detection, and multiplexed immunofluorescence based on cleavable fluorescent dyes (e.g., chemically cleavable or photocleavable dyes), InSituPlex staining. In some embodiments, one or more protein analytes are detected by immunofluorescence microscopy.

[0124] In some embodiments, the method further includes detecting the presence, quantity, and / or localization of one or more RNA analytes within a cell. In some embodiments, one or more RNA analytes are detected by hybridization chain reaction (HCR), fluorescence in situ hybridization (FISH), single-molecule fluorescence in situ hybridization (smFISH), RNAscope, transcriptome-wide or partially transcriptome-wide RNA in situ hybridization-based methods (e.g., MERFISH, seqFISH, or Digital Spatial Profiling), transcriptome-wide or partially transcriptome-wide RNA in situ sequencing methods (e.g., BAR-seq, BOLORAMIS, FISSEQ, Expansion-Seq (ExSeq), or STARmap), and transcriptome-wide or partially transcriptome-wide RNA sequencing methods that preserve some spatial information (e.g., 10xGenomics Visium). In some embodiments, one or more RNA analytes are detected by hybridization chain reaction (HCR).

[0125] II. Manipulated Cells This disclosure also provides engineered cells or populations of engineered cells produced using the methods disclosed herein. In some embodiments, the engineered cells or populations of engineered cells include one or more exogenous promoters integrated into the cell genome. In some embodiments, the population of engineered cells includes an exogenous promoter juxtaposed with an exogenous nucleic acid sequence. In some embodiments, the population of engineered cells includes an exogenous promoter juxtaposed with an endogenous genomic region containing a genomic sequence that is variable among cells in the population. In some embodiments, the exogenous promoter can drive the expression of a genomic sequence that is variable among cells in the population. In some embodiments, one or more exogenous promoters are integrated in a site-specific manner. In some embodiments, one or more exogenous promoters are integrated at random sites in the genome of one or more mammalian cells. In some embodiments, one or more exogenous promoters are integrated into the cell genome by a site-specific nuclease. In some embodiments, one or more exogenous promoters are integrated into the cell genome by a site-specific recombinant enzyme. In some embodiments, the manipulated cell population includes an exogenous promoter juxtaposed with an exogenous nucleic acid that is stably maintained within the cell nucleus but is not integrated into the genome. In some embodiments, the manipulated cell population includes an exogenous promoter juxtaposed with an exogenous nucleic acid sequence that is part of a plasmid or artificial chromosome.

[0126] In some embodiments, the exogenous promoter and the genomic sequence that is variable between cells in a population are separated at 2 kilobases (kb) or less, 1.5 kb or less, 1 kb or less, 900 bp or less, 800 bp or less, 700 bp or less, 600 bp or less, 500 bp or less, 400 bp or less, 300 bp or less, 250 bp or less, 200 bp or less, 150 bp or less, 100 bp or less, 50 bp or less, or 0 bp. In some embodiments, the exogenous promoter and the genomic sequence that is variable between cells in a population are separated at 2 kb or less. In some embodiments, the exogenous promoter and the genomic sequence that is variable between cells in a population are separated at 1.5 kb or less. In some embodiments, the exogenous promoter and the genomic sequence that is variable between cells in a population are separated at 1 kb or less. In some embodiments, the exogenous promoter and the genomic sequence that is variable between cells in a population are separated at 900 bp or less. In some embodiments, the genomic sequence that is variable between the exogenous promoter and cells within a population is separated at a distance of 800 bp or less. In some embodiments, the genomic sequence that is variable between the exogenous promoter and cells within a population is separated at a distance of 700 bp or less. In some embodiments, the genomic sequence that is variable between the exogenous promoter and cells within a population is separated at a distance of 600 bp or less. In some embodiments, the genomic sequence that is variable between the exogenous promoter and cells within a population is separated at a distance of 500 bp or less. In some embodiments, the genomic sequence that is variable between the exogenous promoter and cells within a population is separated at a distance of 400 bp or less. In some embodiments, the genomic sequence that is variable between the exogenous promoter and cells within a population is separated at a distance of 300 bp or less. In some embodiments, the genomic sequence that is variable between the exogenous promoter and cells within a population is separated at a distance of 250 bp or less. In some embodiments, the genomic sequence that is variable between the exogenous promoter and cells within a population is separated at a distance of 200 bp or less. In some embodiments, the genomic sequence that is variable between the exogenous promoter and cells within a population is separated at a distance of 150 bp or less.In some embodiments, the exogenous promoter and the intercellular genomic sequence are separated at a distance of 100 bp or less. In some embodiments, the exogenous promoter and the intercellular genomic sequence are separated at a distance of 50 bp or less. In some embodiments, the exogenous promoter and the intercellular genomic sequence are separated at a distance of 0 bp. In some embodiments, the separation is within the cell's genomic DNA sequence, exon-coding sequence, or RNA sequence. In some embodiments, the separation is within the cell's genome. In some embodiments, the separation is within the exon-coding sequence. In some embodiments, the separation is within the cell's RNA sequence, which includes the exogenous promoter and the intercellular genomic sequence.

[0127] In some embodiments, multiple different exogenous promoters are integrated into the cell's genome. In some embodiments, each exogenous promoter is integrated into a specific site within the cell's genome. In some embodiments, some exogenous promoters are integrated into specific sites within the cell's genome, while others are integrated into random sites within the cell's genome.

[0128] In some embodiments, the exogenous promoter is a sequence-specific RNA polymerase promoter. In some embodiments, the exogenous promoter is a phage promoter. In some embodiments, the exogenous promoter is selected from the group consisting of the T7 promoter, the T3 promoter, and the SP6 promoter. In some embodiments, the exogenous promoter is the T7 promoter. In some embodiments, the exogenous promoter is the T3 promoter. In some embodiments, the exogenous promoter is the SP6 promoter.

[0129] In some embodiments, the manipulated cells include one or more mammalian cells selected from stem cells, mesoderm cells, endoderm cells, ectoderm cells, cardiomyocytes, immune cells, epithelial cells, lung cells, Clara cells, Paneth cells, pancreatic cells, gastric cells, goblet cells, glandular cells, ductal cells, central atrial cells, brush border cells, endocrine cells, thyroid cells, pancreatic islet cells, mucin cells, pituitary cells, neurons, sensory neurons, receptor neurons, neural progenitor cells, pyramidal cells, rod cells, interneurons, astrocytes, oligodendrocytes, ependymal cells, pituitary cells, adipocytes, lipid cells, kidney or urinary tract cells, germ cells, endothelial cells, extracellular matrix cells, contractile cells, skeletal muscle cells, cardiomyocytes, hematopoietic cells, germ cells, nurse cells, or stromal cells. In various embodiments, the manipulated cells include one or more immune cells. In some embodiments, the immune cells are T cells, NK cells, B cells, macrophages, dendritic cells, mast cells, monocytes, neutrophils, basophils, eosinophils, hematopoietic stem cells, or immortalized immune cells. In some embodiments, the immune cells are T cells, NK cells, or B cells. In some embodiments, the immune cells express one or more cell therapy constructs (e.g., engineered immune receptors). In some embodiments, the immune cells encode chimeric antigen receptors (CARs). In some embodiments, the immune cells are chimeric antigen receptor-expressing T (CAR-T) cells or chimeric antigen receptor-expressing NK (CAR-NK) cells. In some embodiments, the immune cells are chimeric antigen receptor-expressing T (CAR-T) cells. In some embodiments, the immune cells are chimeric antigen receptor-expressing NK (CAR-NK) cells. In some embodiments, the biological sample includes embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), or cells derived from ESCs or iPSCs and selectively differentiated into a particular lineage.

[0130] In some embodiments, the population of manipulated cells is homogeneous. In some embodiments, the population of manipulated cells is heterogeneous.

[0131] In some embodiments, the manipulated cells include one or more cells composed of cells derived from a single species. In some embodiments, the manipulated cells include one or more cells derived from multiple species. In some embodiments, the manipulated cells include human cells and mouse cells. In some embodiments, the manipulated cells include human immune cells and mouse cells.

[0132] III. Kit This disclosure also provides kits for carrying out the methods disclosed herein or for preparing engineered cells or engineered cell populations. In some embodiments, the kit includes (a) a sequence-specific RNA polymerase, (b) a reverse transcriptase, (c) a reagent for in situ sequencing, and (d) instructions for using components (a) to (c) to determine in situ the presence, quantity, and / or localization of a nucleic acid sequence of interest in one or more fixed mammalian cells in a biological sample.

[0133] In some embodiments, the sequence-specific RNA polymerase is a phage RNA polymerase. In some embodiments, the sequence-specific RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase. In some embodiments, the sequence-specific RNA polymerase is a T7 promoter. In some embodiments, the RNA polymerase is a T3 RNA polymerase. In some embodiments, the sequence-specific RNA polymerase is an SP6 RNA polymerase.

[0134] In some embodiments, the reverse transcriptase is AMV reverse transcriptase. In some embodiments, the reverse transcriptase is MMLV reverse transcriptase. In some embodiments, the reverse transcriptase is engineered reverse transcriptase.

[0135] In some embodiments, in situ sequencing is synthetic sequencing. In some embodiments, the reagent for synthetic sequencing comprises (i) a plurality of detectably labeled nucleotides and (ii) DNA polymerase. In some embodiments, the detectably labeled nucleotides include cleavable detectable labels. In some embodiments, the detectably labeled nucleotides include reversible strand terminator modifications.

[0136] In some embodiments, in situ sequencing is sequencing by ligation. In some embodiments, the reagent for sequencing by ligation comprises (i) a plurality of detectably labeled oligonucleotides containing degenerate bases, and (ii) a DNA ligase. In some embodiments, the detectably labeled oligonucleotides include cleavable detectable labels. In some embodiments, the detectably labeled oligonucleotides include reversible strand terminator modifications.

[0137] In some embodiments, in situ sequencing is avidity sequencing. In some embodiments, the reagent for avidity sequencing comprises (i) a plurality of detectably labeled avidites, and (ii) a manipulated DNA polymerase. In some embodiments, the detectably labeled avidites include a cleavable detectable label.

[0138] In some embodiments, the kit further includes (i) a DNA primer comprising (A) a 3' nucleic acid sequence complementary to a portion of the target RNA adjacent to the nucleic acid sequence of interest, and (B) a 5' nucleic acid sequence containing a sequence-specific RNA polymerase promoter, or further instructions for designing the DNA primer, and (ii) further instructions for reverse transcribing the target RNA using the DNA primer to generate a cDNA molecule. In some embodiments, the 5' nucleic acid sequence of the DNA primer containing the sequence-specific RNA polymerase promoter is dsDNA. In some embodiments, the dsDNA is hybridized dsDNA or hairpin.

[0139] In some embodiments, the kit further includes (i) reagents for performing second strand synthesis on a cDNA molecule, and / or (ii) further instructions for performing second strand synthesis.

[0140] In some embodiments, the methods provided herein do not involve a DNA degradation step. For example, in some embodiments, the methods do not involve reacting a sample (e.g., any sample or reaction mixture generated during the implementation of the method) with DNase. [Examples]

[0141] The following examples are for illustrative purposes only and are not intended to limit the scope or content of the present invention in any way.

[0142] Example 1: Parameters for Amplifying Variable Barcode Sequences Structure and cell line engineering The construct was designed to include the T7 phage promoter upstream of the target nucleic acid region. In the case of cell lines, the DNA construct was introduced into target cells by lentiviral transduction. To achieve the desired infection multiplicity, lentiviruses were added to mammalian cells, resulting in the integration of the construct into the mammalian cell genome. The tissue samples shown in Figures 4, 5, 6, and 7 were derived from mice with human patient-derived xenograft (PDX) tumors, which were also injected with human CAR T cells. Some of the CAR T cells possessed a T7 promoter (e.g., T7) barcode construct integrated into their genome. The CAR T cells were infiltrating the PDX tumors at the time of tissue preparation. Briefly, tissue samples were prepared using standard FFPE preparation techniques, including fixation with formaldehyde and embedding in paraffin. The paraffin-embedded FFPE blocks were cut into 5 μm thick sections and mounted on glass slides. To perform the described experiments, the tissue samples were first deparaffinized, rehydrated, and antigens recovered.

[0143] RNA transcription from phage promoters in fixed cells or tissues The cells were washed with PBS, fixed, and permeabilized with Triton surfactant. The T7 reaction mixture (buffer, NTP, T7 polymerase, and RNase inhibitor) was added to the cells. The T7 reaction mixture was incubated at a constant, uniform temperature (37°C) for a specific reaction time (6 hours), and RNA was generated from DNA using a phage promoter. Control groups of cell and tissue samples were also prepared without the T7 reaction. After reaction incubation, the cells were washed, and the newly transcribed RNA was fixed in place using formaldehyde.

[0144] Next, the samples were reverse transcribed, and cDNA was generated from the RNA transcript by primer hybridization and treatment with reverse transcriptase. The cDNA was then immobilized in place and used as a target for padlock ligation, gap filling, and ligation to generate a circular DNA template containing the target sequence. The primers were hybridized to the circular DNA, and rolling circle amplification was performed on the circular template to generate many repeating single-stranded DNA (ssDNA) copies of the target sequence. The cells were washed, and RCA amplicons were immobilized. To read the sequence downstream of the T7 promoter, synthetic sequencing was performed by first adding sequencing primers, and then adding fluorescent nucleotides with reversible terminator sequences (allowing the incorporation of only one nucleotide per cycle). The incorporated nucleotides were read in situ using a fluorescence microscope. Figure 3 shows the cell sample, and Figure 4 shows the tissue sample, comparing the T7 reaction sample (lower panel) with the control sample (upper panel) that did not undergo the T7 reaction. DAPI indicates the cell nucleus, while the "T signal" indicates the detected nucleic acid. These results demonstrate that target nucleic acid sequences can be detected in fixed cell samples and FFPEDAPI samples using in-situ transcription and subsequent fluorescent in-situ sequencing.

[0145] In another experiment, tissue samples were prepared as described above. The samples were washed with PBS, fixed, and permeabilized with Triton surfactant. The T7 reaction mixture (buffer, NTP, T7 polymerase, and RNase inhibitor) was added to the cells. The T7 reaction mixture was incubated for 3 hours or 18 hours, and RNA was generated from DNA using a phage promoter. Sequencing by reverse transcription, RCA, and synthesis was performed as described above. Figure 5 shows images of tissue samples incubated with the T7 reaction mixture for 3 hours (top panel) or 18 hours (bottom panel). These results indicate that the signal of the detected barcode nucleic acid increases with increasing T7 reaction duration.

[0146] In another experiment, tissue samples were prepared as described above. Prior to the T7 reaction, cells were treated with RNase to degrade endogenous RNA. The T7 reaction, reverse transcription, RCA, and sequencing by synthesis were performed as described above. Figure 6 shows images of tissue samples treated with RNase (lower panel) or control (upper panel). These results suggest that RNase treatment prior to T7 amplification can improve detection specificity without loss of barcode signals, which may be advantageous.

[0147] In another experiment, tissue samples were prepared, and the T7 reaction, reverse transcription, and RCA were all performed as described above. These samples were then subjected to six consecutive synthetic sequencing and imaging cycles. Figure 7 shows images of DAPI (nucleus), adenine, cytosine, guanine, and thymine channels in the tissue samples after each consecutive round of synthetic sequencing. The results demonstrate that this method generates specific signals in each synthetic sequencing cycle with minimal cross-channel detection.

[0148] Example 2: Improvement of nucleic acid sequence detection by amplification of variable barcode sequences using a promoter incorporated in a biological sample, followed by amplification of the cDNA product. Tissue or cell samples are prepared as in Example 1 above, and a construct without the T7 promoter system is expressed in one group. T7 reaction, reverse transcription, RCA, and synthesis sequencing are performed as in Example 1 above. Images of in situ sequencing results from samples containing the T7 promoter system are compared with images from samples without the T7 promoter system.

[0149] Example 3: Amplification of a variable barcode sequence using a promoter incorporated into a biological sample, followed by amplification of the cDNA product. Structure and cell line engineering In the case of cell lines, the construct is designed to include a target phage promoter (T3, T7, SP6, etc.) upstream of the target nucleic acid region (e.g., a barcode or other variable nucleic acid sequence). The target region may be an endogenous or exogenously introduced sequence. A phage transcription termination sequence is optionally included downstream. In some embodiments, a nucleic acid spacer is included between the promoter and the nucleic acid region.

[0150] The construct, including the tracked promoter and barcode, is genetically introduced into mammalian cells. The DNA construct is introduced into cells by lentivirus. The lentivirus is first produced by incubating a transfer plasmid with a packaging plasmid and envelope plasmid, as well as transfection reagents, and adding it to 293T cells. The lentiviral supernatant is collected from the 293T cells 48 or 72 hours after removal of contaminated cells and stored at -80°C or used immediately. To achieve the desired infection multiplicity, the lentivirus is added to mammalian cells at a specific titer, resulting in the integration of the construct into the mammalian cell genome. Following several days of incubation and cell growth, the percentage of cells expressing the construct is determined by flow cytometry, drug resistance, or other methods. The cells are then grown, passaged, frozen, or used for downstream assays. Cells may also be engineered to contain the construct using other viral vectors, including retroviruses or AAVs, or other methods such as gene editing.

[0151] RNA transcription from phage promoters in fixed cells or tissues For fixed cells, wash the cells with PBS, fix them, and permeabilize them with Triton surfactant. Add the T7 reaction mixture to the cells (the T7 reaction mixture contains buffer, NTP, T7 polymerase, and RNase inhibitor). Incubate the T7 reaction mixture at a constant, uniform temperature (37-42°C) for a specified reaction time (3-18 hours) to generate RNA from DNA using a phage promoter. After reaction incubation, wash the cells and fix the newly transcribed RNA in place (e.g., using formaldehyde).

[0152] Next, the sample undergoes reverse transcription, and DNA is generated from the RNA transcript by primer hybridization and treatment with reverse transcriptase. Then, the cDNA is immobilized in place (e.g., using formaldehyde or glutaraldehyde), and all or part of the complementary RNA is optionally digested. The cDNA is then used as a target for padlock junction, gap filling, and ligation to generate a circular DNA template containing the variable sequence. The primers are hybridized to the circular DNA, and rolling circle amplification is performed on the circular template to generate many ssDNA copies of the target nucleic acid region. The cells are washed, and the RCA amplicon is immobilized. Synthetic sequencing is performed by first adding sequencing primers to read the barcode sequence downstream of the T7 promoter, followed by adding fluorescent nucleotides with reversible terminator sequences (allowing the incorporation of only one nucleotide per cycle). The incorporated nucleotides are read in situ using a fluorescence microscope. After imaging in each step, the fluorophores and reversible terminators are cleaved, and the sequencing process is repeated by synthesis to obtain the desired barcode sequence.

[0153] For tissues, tissue samples containing cells with promoter (e.g., T7) barcode constructs are first deparaffinized, rehydrated, and subjected to antigen recovery. The tissue then undergoes the workflow described above.

[0154] Example 4: Amplification of variable barcode sequences using a promoter embedded in a biological sample without amplification of cDNA products. Structure and cell line engineering The construct is designed to include a target phage promoter (T3, T7, SP6, etc.) upstream of a target nucleic acid region (e.g., a barcode or other variable nucleic acid sequence). The target region may be an endogenous or exogenously introduced sequence. A phage transcription termination sequence is optionally included downstream. In some embodiments, a nucleic acid spacer is included between the promoter and the nucleic acid region.

[0155] The construct, including the tracked promoter and barcode, is genetically introduced into mammalian cells. The DNA construct is introduced into cells by lentivirus. The lentivirus is first produced by incubating a transfer plasmid with a packaging plasmid and envelope plasmid, as well as transfection reagents, and adding it to 293T cells. The lentiviral supernatant is collected from the 293T cells 48 or 72 hours after removal of contaminated cells and stored at -80°C or used immediately. To achieve the desired infection multiplicity, the lentivirus is added to mammalian cells at a specific titer, resulting in the integration of the construct into the mammalian cell genome. Following several days of incubation and cell growth, the percentage of cells expressing the construct is determined by flow cytometry, drug resistance, or other methods. The cells are then grown, passaged, frozen, or used for downstream assays. Cells may also be engineered to contain the construct using other viral vectors, including retroviruses or AAVs, or other methods such as gene editing.

[0156] RNA transcription from phage promoters in fixed cells or tissues For cell samples, the cells are washed with PBS, fixed, and permeabilized with Triton surfactant. The T7 reaction mixture is added to the cells (the T7 reaction mixture contains buffer, NTP, T7 polymerase, and RNase inhibitor). The T7 reaction mixture is incubated at a constant, uniform temperature (37-42°C) for a specified reaction time (3-18 hours) to generate RNA from DNA using a phage promoter. After reaction incubation, the cells are washed and the newly transcribed RNA is fixed in place (e.g., using formaldehyde).

[0157] Next, the sample is reverse transcribed, and DNA is generated from the RNA transcript by primer hybridization and treatment with reverse transcriptase. The primers contain one region (3' end) complementary to the RNA and another region (5' end) containing the T7 promoter sequence. After reverse transcription, the cDNA is optionally fixed in place (e.g., using formaldehyde or glutaraldehyde), and all or part of the complementary RNA is optionally digested. To read the barcode sequence from the cDNA product, synthetic sequencing is performed directly by first adding a sequencing primer, and then adding a fluorescent nucleotide with a reversible terminator sequence (allowing the incorporation of only one nucleotide per cycle). The incorporated nucleotide is read in situ using a fluorescence microscope. After imaging in each step, the fluorophore and reversible terminator are cleaved, and the synthetic sequencing process is repeated to obtain the desired barcode sequence.

[0158] For tissue samples, tissue samples containing cells with promoter (e.g., T7) barcode constructs are first deparaffinized, rehydrated, and subjected to antigen recovery. The tissue then undergoes the workflow described above.

[0159] Example 5: Amplification and sequencing of endogenous or exogenous sequences without an integrated promoter - Second-strand synthetic version Cells or tissues containing the target RNA (either endogenously or exogenously introduced) are fixed or processed as described above.

[0160] For fixed cells, the cells are washed with PBS, fixed, and permeabilized with Triton surfactant. The sample is reverse transcribed, and DNA is generated from the endogenous RNA transcript by primer hybridization and treatment with reverse transcriptase. The primers contain one region (3' end) complementary to the RNA and another region (5' end) containing the T7 promoter sequence. After reverse transcription, the cDNA is optionally fixed in place (e.g., using formaldehyde or glutaraldehyde), and all or part of the RNA is optionally digested. Second strand synthesis is performed using primers that hybridize to the cDNA such that, at the end of second strand synthesis, both the T7 promoter and the target nucleic acid region are double-stranded, and the T7 promoter drives transcription of the target region.

[0161] The T7 reaction mixture is added to the cells (the T7 reaction mixture contains buffer, NTP, T7 polymerase, and an RNase inhibitor). The T7 reaction mixture is incubated at a constant, uniform temperature (37-42°C) for a specified reaction time (3-18 hours) to generate RNA from DNA using a phage promoter. After reaction incubation, the cells are washed and the newly transcribed RNA is immobilized in place (e.g., using formaldehyde). A second RT reaction is performed on these RNA transcripts according to the examples outlined above, and in-situ sequencing is performed using rolling circle amplification (e.g., according to Example 1 above) or directly (e.g., according to Example 2 above).

[0162] Amplification and sequencing of endogenous or exogenous sequences without integrated promoters - double-stranded primers Cells or tissues containing the target RNA (either endogenously or exogenously introduced) are fixed or processed as described above.

[0163] For cell samples, cells are washed with PBS, fixed, and permeabilized with Triton surfactant. The sample is reverse transcribed, and DNA is generated from the endogenous RNA transcript by primer hybridization and treatment with reverse transcriptase. The primers consist of one region (3' end) complementary to the RNA and another region (5' end) containing a double-stranded T7 promoter sequence (e.g., a single oligo, a hairpin from two short oligos linked together, or other configurations to double-strand a portion of the T7 promoter region). After reverse transcription, the cDNA is optionally fixed in place (e.g., using formaldehyde or glutaraldehyde), and the RNA is optionally digested. The T7 reaction mixture is added to the cells (the T7 reaction mixture contains buffer, NTP, T7 polymerase, and an RNase inhibitor). The T7 reaction mixture is incubated at a constant, uniform temperature (37-42°C) for a specified reaction time (3-18 hours), and RNA is generated from the DNA using a phage promoter. After reaction incubation, the cells are washed and the newly transcribed RNA is immobilized in place (e.g., using formaldehyde). A second RT reaction is performed on these RNA transcripts according to the examples outlined above, and in situ sequencing is performed using rolling circle amplification (e.g., according to Example 2 above) or directly in the previous step (e.g., according to Example 3 above).

[0164] The methods provided herein offer surprising and unexpected results compared to previous methods. For example, U.S. Patent No. 11,421,273 and AKSary et al. Nat Biotechnol 38,66-75 (2020), “Zombie” (Zombie is an optical measurement of barcodes by in-situ expression) provide a method for in-situ reading of DNA barcodes via in vitro transcription, but such methods have limitations, such as being able to detect only barcodes of at least 20 nucleotides in length. This is in contrast to the methods provided herein. Zombie also has drawbacks, as it requires treatment with DNase that degrades the DNA in the sample, thus hindering further probing of the target DNA and making it unsuitable for formalin-fixed samples. Therefore, the embodiments and examples provided herein overcome not only these problems but also other problems that other methods have.

[0165] Example 6: Amplification of a variable barcode sequence using a promoter incorporated into a biological sample, followed by amplification of a cDNA product. Structure and cell line engineering An example of this workflow is shown in Figure 8. The construct was designed to include a target promoter (e.g., T3, T7, SP6, etc.) upstream of the target nucleic acid sequence or region (e.g., a barcode or other variable nucleic acid sequence). The target sequence was either an endogenous or exogenously introduced sequence. An optional phage transcription termination sequence was included downstream. In some cases, a nucleic acid spacer was inserted between the promoter and the target nucleic acid sequence.

[0166] DNA constructs containing tracked phage promoters and barcodes were genetically introduced into mammalian cells by lentiviral transduction. Lentiviruses were produced by incubating the construct-containing transfer plasmid with a packaging plasmid, an envelope plasmid, and a transfection reagent, and then adding this transfection mixture to 293T cells. The lentiviral supernatant was collected from the 293T cells 48 or 72 hours after removal of contaminated cells. The lentiviruses were then stored at -80°C or used immediately. To achieve the desired infection multiplicity, lentiviruses were added to mammalian cells at specific titers, resulting in the integration of the constructs into the mammalian cell genome. In some examples, the mammalian cells transduced with lentiviruses were human primary T cells. In some cases, lentiviral transduction resulted in T cells expressing one or more chimeric antigen receptors (CARs), endogenous human proteins, variants or truncated forms of human proteins, synthetic proteins, proteins derived from other organisms, non-coding RNAs such as short hairpin RNA (shRNA), or combinations of one or more of these protein or RNA classes. Following several days of incubation and cell growth, the percentage of cells expressing the construct was determined by flow cytometry, drug resistance, or other methods. The cells were then grown, passaged, frozen, or used in downstream assays. Cells containing the construct could also be manipulated using retroviruses or other viral vectors, including AAV, or by other methods such as gene editing.

[0167] Preparation of mouse models and tissue samples Genetically modified cells containing constructs including phage promoters and barcodes were prepared for injection into a mouse cancer model by washing the cells with PBS and resuspending them to the desired injection concentration. 1 × 10 6 ~10×10 6Human T cells were injected into immunosuppressed NOD-scid gamma (NSG) mice that had previously received human patient-derived xenograft (PDX) tumors or human cancer cell line-derived xenograft (CDX) tumors. Tissue samples shown in Figures 9A-9C and 24A-24F were from mice with orthotopic gastric PDX tumors, tissue samples shown in Figures 10A-10C and 11A-11C were from mice with Hep G2 CDX tumors, and tissue samples shown in Figures 12A-12C and 13A-13C were from mice with AsPC-1 CDX tumors. For these tissue samples, mice were injected with a library of human CAR T cells, the CAR design construct was incorporated into the T cell genome, and the phage promoter was included upstream of the variable barcode. Tumor tissue samples were collected at different time points after CAR T cells had infiltrated the tumor and prepared using standard FFPE preparation techniques. In short, mice were euthanized, the tumors were dissected, fixed with formaldehyde or formalin, and embedded in paraffin. The paraffin-embedded blocks were cut into 5 μm thick sections and mounted on glass slides.

[0168] RNA transcription from phage promoters in fixed cells or tissues Tissue samples prepared by the above FFPE were deparaffinized, rehydrated, and antigens were recovered. Optionally, tissue samples were bleached, and endogenous background autofluorescence signals were reduced using hydrogen peroxide and light. For in vitro cell samples, cells were washed with PBS, fixed, and permeabilized. For both tissue and in vitro cell samples, a T7 (or other phage polymerase) reaction mixture (buffer, rNTP, phage polymerase, and RNase inhibitor) was added to the sample, and the reaction mixture was incubated with the sample at a constant, uniform temperature (37–42°C) for the specified reaction time (3–18 hours) to generate RNA from DNA using a phage promoter. After reaction incubation, the samples were washed, and the newly transcribed RNA was fixed in place (e.g., using formaldehyde).

[0169] Next, tissue or cell samples were reverse transcribed, and cDNA was generated from the RNA transcript by primer hybridization and treatment with reverse transcriptase. The generated cDNA was then immobilized in place (e.g., using formaldehyde or glutaraldehyde), and all or part of the complementary RNA was optionally digested. The cDNA was then used as a target for DNA padlock binding, gap filling, and ligation to the 5' and 3' sequences of the barcode to generate a circular DNA template containing the target nucleic acid sequence. The primers were hybridized to the circular DNA, and rolling circle amplification was performed on the circular template to generate many ssDNA copies of the target nucleic acid sequence. The tissue or cell samples were washed, and the ssDNA amplicons were immobilized. To read the barcode sequence downstream of the T7 promoter, synthetic sequencing (SBS) was performed by first adding sequencing primers, and then adding fluorescent nucleotides with reversible terminator sequences to allow incorporation of only one nucleotide per round. After each imaging step, the fluorophores and reversible terminators were cleaved from the sample, and the SBS process was repeated to obtain the entire barcode sequence of the desired type. Tissue or cell samples were stained with DAPI to mark the cell nuclei.

[0170] In each round of SBS, the fluorescent nucleotide signal was imaged and read out in situ by fluorescence microscopy, and the DAPI signal was detected using one fluorescent channel per nucleotide and one fluorescent channel. Fields of view for FFPE tissue section images were selected to cover all or part of the tissue section, and fields of view for in vitro images were selected to cover all or part of the wells from a 96-well plate. Fields of view were partially overlapping in some cases to allow downstream computational image stitching.

[0171] Raw microscope images were first subjected to illumination correction. Illumination correction was performed individually for each channel and each round of acquisition, as shading artifacts can vary between channels and acquisition rounds. Overlap tiles were aligned for stitching using nuclear images and a phase cross-correlation approach from the scikit-image Python package. Images of entire slides obtained from different rounds were aligned to a reference round using the scikit-image SIFT algorithm for keypoint detection and RANSAC for transformation estimation. Nuclear and cell segmentation was performed using nuclear and actin-stained images, respectively. Segmentation was performed using a convolutional neural network approach specifically trained on tissue images, followed by a watershed algorithm. Barcode detection consisted of several steps. First, background noise was removed by applying Laplacian of Gaussian filters of different sizes to each image. Next, the images were symmetrically arranged and resized before spot detection. Spots were filtered based on filtered and raw mass to remove faint or small spots caused by artifacts. Subsequently, the spots were mapped across channels, and spots present with similar intensity across multiple channels were removed. The remaining spots were mapped to cells, and finally, the complete barcode was reconstructed using cell identification and fuzzy joining for mapping positions between rounds, allowing for shifts of up to 3 pixels in the x and y directions. The barcode of the present invention can be designed so that the minimum Hamming distance is 3, thereby allowing for one mismatch or one missing round in barcode identification.

[0172] In another experiment, samples were prepared from multiple tissue types. The tissues were prepared and treated with T7 reaction, reverse transcription, cDNA amplification, and SBS as described above. Figures 9A-9C show tissue samples from mice with orthotopic gastric PDX tumors, Figures 10A-10C and 11A-11C show tissue samples from mice with Hep G2 CDX tumors, and Figures 12A-12C and 13A-13C show tissue samples from mice with AsPC-1 CDX tumors. In Figures 9A-9C, mice were injected with CAR T cell libraries of seven unique CAR designs, each with its own unique barcode. In Figures 10A-10C, mice were injected with CAR T cell libraries of nine unique CAR designs, each with its own unique barcode.

[0173] These results demonstrate that the presence and localization of target sequences can be detected from multiple in vivo sample types using T7 amplification followed by an in-situ sequencing system. The quantification of barcodes detected from these tissue samples using the methods detailed above is shown in Figures 14A–14C and Table 1 (both corresponding to the samples in Figures 10A–10C), Figure 15 and Table 2 (both corresponding to the samples in Figures 9A–9C), Figures 16A, 16B, and Table 3 (both corresponding to the samples in Figures 11A–11C), Figures 17A, 17B, and Table 4 (both corresponding to the samples in Figures 12A–12C), and Table 5 (corresponding to the samples in Figures 24A–24F). The quantifications in these figures and tables demonstrate the high level of detection of the presence and number of barcodes contained in tissue samples using the T7 amplification and SBS readout method described above.

[0174] Table 1 shows the barcode detection specificity of the expected barcodes present in the tissue samples. A library of nine chimeric antigen receptor (CAR) designs, each with a unique barcode following the T7 promoter, was introduced into mice with pre-transplanted HepG2 tumors, and these tumors were then extracted to generate FFPE tissue sections. Sixty negative control barcodes, indicated as barcode holdout (BH) designs 1–59, including either design 9A or 9B, were not present in any construct within the library. As shown in the table, unique CAR designs 1–8 were part of the 9mer libraries of all three donors, while designs 9A and 9B had the same CAR design but different barcodes; design 9A was present only in the 9mer libraries of donors 1 and 2, and design 9B was present only in the 9mer library of donor 3. In summary, the majority of barcode holdouts were not highly detectable in FFPE tissue sections, but barcodes that were part of the nine CAR libraries were detected more frequently in T cells of all three donors. [Table 1-1] [Table 1-2]

[0175] Table 2 shows the barcode detection of a library of seven CAR designs, each with a unique barcode following the T7 promoter. The pool of seven CAR designs was injected into mice with patient-derived xenograft tumors orthotopically implanted in the stomach, and the tumors were then excised and processed into FFPE tissue sections. All seven barcoded designs were successfully detected. [Table 2]

[0176] Table 3 shows the specificity and scalability of in vivo barcode detection from tissue samples. A library of 56 CAR designs, each with a unique barcode following the T7 promoter, is shown as design numbers 1-56. Thirteen barcode holdouts are negative control barcoded constructs not included in the library. The barcoded CAR library was transduced into T cells and injected into a xenograft tumor mouse model derived from the HepG2 cell line to generate FFPE tumor sections. Barcodes including those in the 56 CAR design libraries were highly detected, while barcode holdouts were poorly detected or not detected at all. [Table 3-1] [Table 3-2] [Table 3-3]

[0177] Table 4 shows barcode detection for a library of 80 CAR designs, each with a unique barcode following the T7 promoter, from FFPE tissue sections of xenografts derived from the AsPC-1 cell line. The designs include structural modifications to CARs (CAR numbers 1–17), logic gate CARs (logic gate numbers 1–2), and armored CARs (Armor numbers 1–61). Designs for each of these classes (structural modifications of CARs, logic gate CARs, and armored CARs) were detected. Barcodes that were not detected may indicate constructs with CAR designs that adversely affect T cell function or compatibility within the tumor. [Table 4-1] [Table 4-2] [Table 4-3]

[0178] Table 5 shows barcode detection of a library of seven uniquely barcoded CAR designs derived from FFPE tissue sections from xenograft mouse tumors from orthotopic gastric patients. The CAR library contained six constructs with a barcode after the T7 promoter and one construct with a barcode after both the T7 and SP6 promoters. The columns in the table, from left to right, are: design number, design barcode read using SBS followed by the phage polymerase system, whether a particular design contained a barcode after the T7 promoter, whether a particular design contained a barcode after the SP6 promoter, the number of each barcode design detected in FFPE tissue sections treated with T7 for barcode amplification, and the number of each barcode design detected in FFPE tissue sections treated with T7 for barcode amplification. The results in this table indicate that both T7 and SP6 phage polymerases efficiently and specifically amplify nucleic acid barcode signals from constructs containing the relevant promoters. This demonstrates that the phage promoter barcode amplification system used here is not limited to a single phage polymerase enzyme; that is, multiple polymerases can be used to amplify sequences from multiple unique constructs within the same target sample or across different target samples. [Table 5]

[0179] In another experiment, barcodes were detected from libraries of multiple DNA construct types. Here, constructs encoded designs that encoded different proteins or non-proteins, and each construct also contained a variable nucleic acid barcode downstream of the T7 promoter. Tissue samples were prepared and subjected to T7 reaction, reverse transcription, cDNA amplification, and sequencing by synthesis for barcode detection as described above. Figures 10A–10C illustrate tissue samples containing CAR T cells prepared from a DNA construct library containing nine chimeric antigen receptors (CARs), including armored and unarmored CARs. The nine unique barcoded CAR designs in the libraries derived from the cells shown in Figures 10A–10C are listed in Table 1 and quantified in Figures 14A–14C. Figures 9A–9C illustrate tissue samples containing CAR T cells prepared from a DNA construct library containing seven CARs. The seven unique barcoded CAR designs in the libraries derived from the cells shown in Figures 9A–9C are listed in Table 2 and quantified in Figure 15. Figures 11A-11C illustrate tissue samples containing CAR T cells prepared from a DNA construct library containing 56 CARs, including armored and unarmored CARs. The 56 unique barcoded CAR designs in the library derived from the cells shown in Figures 11A-11C are listed in Table 3 and quantified in Figures 16A and 16B. Figures 12A-12C illustrate tissue samples containing CAR T cells prepared from a DNA construct library containing 80 CAR designs, including structurally different unarmored CARs, armored CARs, and logic gate CARs. The 80 unique barcoded CAR designs in the library derived from the cells shown in Figures 12A-12C are listed in Table 4 and quantified in Figures 17A and 17B. Figures 13A-13C illustrate tissue samples containing CAR T cells prepared from a DNA construct library containing 12 CARs, each containing one unarmored CAR and 11 CARs co-expressed with different shRNAs. These results demonstrate that the presence and number of multiple types of protein-coding and non-protein-coding DNA construct libraries derived from tissue samples can be determined using T7 barcode amplification followed by in-situ SBS.These results also demonstrate that the T7 barcode amplification system can be used to detect and quantify target sequences from DNA construct libraries, regardless of the number of constructs included in the library.

[0180] In another experiment, cells were used in vitro after gene integration of the phage promoter and barcode-containing constructs described above, without mouse injection or tissue preparation. Cells were washed with PBS, fixed, and permeabilized. T7 reactions, reverse transcription, cDNA amplification, and SBS were performed as described above. Figures 18A, 18B, 19A, and 19B illustrate in vitro T cells with CAR design constructs containing a T7 promoter upstream of the variable barcode. Figure 20 shows the quantification, and Table 1 lists nine uniquely barcoded CAR designs from the in vitro T cell library shown in Figures 18A and 18B. Figure 21 illustrates the quantification from in vitro barcode detection of seven uniquely barcoded CARs, listed in Table 2, showing that all barcoded designs were highly detected in vitro. These results demonstrate that the barcode or nucleic acid sequence of interest can be detected and quantified from both in vitro cell and in vivo tissue samples using T7 amplification and subsequent in situ SBS. The provided method is advantageous for drug candidate screening because it allows for the simultaneous screening of multiple drug candidates in a single sample. The provided method also offers enhanced detection of nucleic acid sequences in situ.

[0181] Example 7: Detection of variable barcode sequences is accurate. The accuracy of barcode detection was determined by comparing the detection of barcoded DNA constructs known to be present in tissue samples with the detection of barcoded DNA constructs not present in tissue samples (referred to as "barcode holdouts"). In this example, a library of nine CAR constructs was designed, each possessing a unique barcode downstream of the T7 promoter. Sixty negative control CAR constructs were also designed to include barcodes and T7 promoters, but these constructs were excluded from the library. The nucleotide sequences of the barcode holdouts and the barcodes on the nine CAR libraries were designed using the same design requirements, including avoiding homopolymer sequences. The nine construct CAR libraries were transduced into T cells isolated from one of three different human lymphocyte donors using the approach described in Example 6 above. The construct-containing cells were then introduced into mice pre-transplanted with Hep G2 CDX tumors, as described in Example 6. Tissue sections were prepared and subjected to T7 amplification, followed by reverse transcription, cDNA amplification, and in-situ SBS, as described in Example 6. The number of barcoded constructs present in samples containing cells derived from three donors was determined. Figures 14A–14C and 22A–22B illustrate the quantification of barcodes detected in vivo from tissue samples containing transduced T cells with nine barcoded CAR libraries. Table 1 lists the CAR design, nucleotide barcode reading using the T7 system, whether each barcoded CAR construct was introduced into T cells derived from each of the three donors, and the number of barcodes detected from these samples. The number of detected barcodes was compared between the nine CAR designs introduced into the samples (true barcodes) and 60 negative control designs called barcode holdouts (BHs) that were not included in the construct library introduced into the samples. Barcodes from the nine introduced CAR designs were detected in large numbers in the samples, while barcode holdouts were detected in small numbers or not detected at all.The accuracy of this detection by the T7 amplification system of barcoded constructs contained in tissue, compared to barcode holdout, was observed in samples containing cells prepared from all three donors.

[0182] To further establish accuracy, barcoded CAR designs 9A and 9B were designed to contain the same CAR but different barcodes. Design 9A was included only in libraries introduced into cells from donors 1 and 2, while design 9B was included only in libraries introduced into cells from donor 3. Barcode detection results showed that CAR designs 1-8, common to cells from all three donors, were highly detected in cell tissue samples from all three donors, while design 9A was highly detected only in cells from donors 1 and 2, and design 9B was highly detected only in cells from donor 3. In particular, the detection of an equal number of barcodes from each design was not expected, as the CAR designs in the nine CAR libraries were expected to affect the number of cells with each design in the tissue, independently of the barcodes. Overall, these results demonstrate that T7 promoter-based amplification, as well as detection by cDNA amplification and SBS, accurately distinguish DNA constructs in in vivo samples based on the unique identifying nucleotide sequences of the barcodes contained in each construct.

[0183] In another experiment, the accuracy of barcode detection was also demonstrated from tissue samples containing T cells transduced with a library of 56 barcoded CAR constructs generated, processed, and barcode amplified as described above. Figures 16A and 16B show a quantification comparing the number of barcodes detected between the 56 CAR designs and 13 barcode holdouts included in the sample. Table 3 lists the CAR design number, the design name from Figures 16A and 16B, the nucleotide barcode readout using the T7 system, whether each barcoded CAR construct was included in the pool of 56 CAR libraries introduced into the cell sample, and the number of barcodes detected from the tissue sample containing these cells. Barcode detection from tissue samples using the 56 CAR T cell library demonstrates high detection of barcoded constructs that were part of the library, compared to no detection or only slight detection of barcode holdouts. As explained for the nine CAR libraries above, the different CAR designs, including the 56 CAR libraries, were expected to affect the number of cells with each design in the tissue, independently of the barcodes; therefore, the detection of the same number of barcodes from each design was not expected. Overall, these results demonstrate that the phage promoter system for in vivo nucleic acid amplification and detection by cDNA amplification and SBS has very high accuracy for the design constructs and barcodes contained in the libraries. This accuracy improves the detection and quantification of nucleic acid species in situ and enables simultaneous testing of multiple constructs with unique barcodes in the sample.

[0184] Example 8: Amplification of variable barcode sequences in a biological sample using a T7 or SP6 promoter, followed by amplification of the cDNA product. Tissue samples were prepared as in Example 6 and subjected to in vivo barcode amplification using either T7 or SP6 RNA polymerase. To generate the tissue samples, T cells were transduced in a library of seven unique barcode CAR designs. The CAR library contained six constructs with a T7 RNA polymerase promoter upstream of the nucleic acid barcode, and one construct (T7+SP6 promoter construct) with both a T7 RNA polymerase promoter and an SP6 RNA polymerase promoter upstream of the nucleic acid barcode. T cells containing either the T7 promoter construct or the T7+SP6 promoter construct were injected into an orthotopic gastric PDX tumor mouse model, and tissue samples were prepared as described in Example 6. Barcode amplification was performed on the tissue samples using either the T7 RNA polymerase reaction or the SP6 RNA polymerase reaction, as well as subsequent reverse transcription, cDNA amplification, and in-situ SBS. Figure 23 shows a schematic diagram illustrating experiments comparing barcode detection using either the T7 or SP6 promoter. The uniquely barcoded CAR library was designed to contain six constructs with barcodes after the T7 promoter and one construct with barcodes after both the T7 and SP6 promoters. The barcoded construct library was transduced into CAR T cells and injected into a mouse model. The mice were euthanized, tissue was collected, and the barcodes were amplified using either T7 or SP6 phage polymerase, followed by reverse transcription, cDNA amplification, and in-situ SBS. Post-SBS tissue imaging identified barcode signals from all library constructs in the T7-treated samples, while only the SP6 promoter-containing construct was identified in the SP6-treated samples. Figures 24A–24F illustrate tissue samples containing CAR T cells prepared from seven CAR libraries with barcodes detected using either the T7 or SP6 promoter system. The T7 amplification system detected barcodes from all seven T7 promoters containing the CAR designs, while the SP6 amplification system highly detected barcodes only from the CAR designs containing the SP6 promoter.This example demonstrates the specificity of a phage promoter amplification system for detecting target nucleic acid sequences only from constructs containing phage promoters recognized by RNA polymerase enzymes, which is consistent with the sequence specificity of these enzymes. Table 5 shows the quantification of the number of barcodes detected from tissue samples shown in Figures 24A–24F using either the T7 or SP6 barcode amplification method. The number of barcodes from constructs containing T7 and SP6 promoters was similar between tissue samples treated with T7 polymerase and SP6 polymerase, with 694 and 728 barcodes detected, respectively. This result indicates that both T7 and SP6 polymerases efficiently amplify nucleic acid barcode signals in constructs containing the relevant promoters. This result also demonstrates that the phage promoter and SBS detection system is generalizable and not specific to a single RNA polymerase enzyme or promoter, and that other enzymes and promoters can be used in the method provided herein.

[0185] Example 9: Amplification of multiple nucleic acid sequences from multiple phage promoters within a single cell. A phage promoter barcode amplification system was used to distinguish multiple target nucleic acid sequences from a single cell. In this example, 16 CAR design constructs were generated, each having a unique barcode following the T7 promoter. Lentiviruses were then transduced into T cells with multiple infection degrees, introducing 0, 1, 2, or more constructs per individual cell. The cells were fixed in vitro and subjected to transcription, reverse transcription, cDNA amplification, and in-situ SBS driven by the T7 promoter, as described in Example 6. Barcode detection was performed to determine the number and identity of CAR DNA constructs contained in each cell. Figures 19A and 19B illustrate microscopic images of T cells transduced with the CAR library. Cells shown as rhombic and circular contain two uniquely barcoded CAR constructs, while cells shown as square contain a single uniquely barcoded CAR construct. These results demonstrate that multiple target nucleic acid sequences, each downstream of a phage promoter, can be detected and distinguished from a single cell using a phage amplification system.

[0186] Example 10: Amplification of variable barcode sequences using promoters embedded in in vivo tissue samples, and combination of amplification of cDNA products and protein detection. The construct design, cell line manipulation, mouse model, and FFPE tissue section processing were carried out as described in Example 6 above. To combine protein detection with nucleic acid sequence amplification using the T7 promoter, tissue sections were deparaffinized, rehydrated, and antigen recovered. The tissue was incubated in blocking buffer to reduce nonspecific antibody binding, and then washed with PBS. Tissue sections were incubated overnight with either a primary antibody or a fluorophore-conjugated antibody. After incubation with the primary antibody, the tissue sections were washed and optionally incubated with a fluorophore-conjugated secondary antibody. DAPI staining was performed to mark cell nuclei, and tissue sections were imaged to detect protein signals from the antibody-stained tissue. In some examples, the tissue was repeatedly stained to detect additional proteins using antibodies by removing fluorophores through rigorous washing and additional multiple antibody stainings performed as described above. Next, tissue sections were washed with formamide / SSC and PBS, and barcode amplification was performed using the approach described in Example 6, including T7 promoter transcription, reverse transcription, cDNA amplification, in-situ SBS, and imaging for barcode detection. For some samples, antibody-based tissue staining was performed after the barcode detection process was completed to detect proteins. Figures 25A–25D illustrate microscopic images derived from tissue sections from a HepG2 CDX tumor mouse model containing transduced T cells with a library of 56 uniquely barcoded CAR constructs. Tissue sections were subjected to barcode amplification with T7 RNA polymerase, followed by reverse transcription, cDNA amplification, and in-situ SBS. Protein analytes CD8, granzyme B, LAG3, and PDL1 were stained before T7 amplification and barcode detection by SBS, and cytokeratin was stained after T7 amplification and barcode detection by SBS. Before euthanizing the mice and harvesting the tumors, the mice were injected with a library of 56 uniquely designed CAR T cells, including both armored and unarmored CAR constructs, each with its own unique barcode.Figures 26A–26D illustrate microscopic images derived from tissue sections of an AsPC-1 CDX tumor mouse model containing transduced T cells with a library of 80 uniquely barcoded CAR constructs. Tissue sections were subjected to barcode amplification with T7 RNA polymerase, followed by reverse transcription, cDNA amplification, and in-situ SBS. Protein analytes CD8, granzyme B, and LAG3 were stained before barcode detection by T7 amplification and SBS, while cytokeratin was stained after barcode detection by T7 amplification and SBS. Before euthanasia and tumor harvesting, mice were injected with a library of 80 uniquely designed CAR T cells containing both armored and unarmored CAR constructs, each with its own unique barcode. Tissue sections shown in Figures 25A–25D and 26A–26D were subjected to T7-driven barcode amplification and antibody staining for protein markers. Antibody signals from staining of T cell-specific proteins, including CD8, LAG3, and granzyme B, were specifically localized to T cells and not strongly detected in cancer cells. Antibody signals from staining of T tumor cell-specific proteins, including cytokeratin and PD-L1, were specifically localized to target cancer cells and not strongly observed in T cells. Signals from both in-situ barcode amplification and T cell-specific protein antibody staining were detected within CAR-containing T cells. This demonstrates that the T7 amplification system can be used to identify and quantify barcoded constructs, including cells, in combination with the detection of physiologically relevant proteins. Signals from in-situ barcode amplification in these tissue samples were detected more strongly in T cells than in cancer cells, and further demonstrated that nucleic acid barcodes amplified by the T7 system were specifically found within cells into which the constructs were introduced. Figure 27 illustrates the quantification of the percentage of cells in which T7-amplified barcoded constructs were detected, defined as positive or negative for CD45 antibody staining, in FFPE tissue samples. This sample contained cells transduced with the 80 CAR libraries listed in Table 4.The tissue underwent barcode detection via amplification based on the T7 promoter, as described above, and was further stained with protein using an antibody against CD45, which is endogenously expressed by T cells. CD45-positive cells represent T cells, while CD45-negative cells represent other cell types that are not T cells, including AsPC-1 cancer cells. Barcodes were detected more strongly in CD45-positive cells compared to CD45-negative cells. This indicates that the T7 system for nucleic acid amplification specifically identified intracellular barcodes, including barcoded CAR constructs.

[0187] Example 11: Amplification of variable barcode sequences and cDNA product amplification using a promoter integrated into an in vivo tissue sample, combined with RNA detection. The construct design, cell line manipulation, mouse model preparation, and FFPE tissue section processing were carried out as described in Example 6 above. To combine RNA detection with nucleic acid sequence amplification using the T7 promoter, the tissue sections were deparaffinized, rehydrated, antigen recovered, and washed with PBS. The tissue sections were then subjected to a hybridization chain reaction (HCR), and the samples were first pre-hybridized by incubation with a hybridization buffer containing formamide and SSC. Single-stranded DNA HCR probes having sequences specific to binding to RNA transcript target sequences (EGFR and WPRE) were added to the tissue samples in the hybridization buffer and incubated overnight to allow the probes to anneal to the target RNA. The tissue was washed with SSC to remove nonspecifically bound HCR probes, and an HCR DNA hairpin amplifier tethered to a fluorophore was added to the sample in the amplification buffer. The tissue sections were incubated with the amplifier overnight, and excess HCR hairpins were removed by washing with SSC and PBS. Tissues were stained with DAPI and imaged for RNA target-specific fluorescent HCR signals. To amplify and detect T7 barcodes, the HCR signal was removed from the tissue by RNase treatment, and then, as described in Example 6, samples underwent T7 promoter transcription, reverse transcription, cDNA amplification by rolling circle amplification, in-situ SBS using sequencing primers, imaging, and barcode detection. Figures 28A–28D illustrate microscopic images of tissue sections from a Hep G2 CDX tumor mouse model containing T cells transduced with a library of 56 uniquely barcoded CAR constructs. The illustrated tissues underwent HCR amplification to amplify RNA from the barcoded CAR constructs, followed by T7-driven barcode amplification. In the illustrated samples, signals from both in-situ barcode amplification and HCR amplification of CAR RNA were detected within the CAR-containing T cells. This demonstrates the specificity of the T7 system for detecting barcodes in cells expressing barcoded construct RNA.

[0188] In summary, the examples and methods provided herein demonstrate the ability to detect and analyze one or more nucleic acid sequences of interest based on the presence, abundance, and localization of constructs, through a combination of the steps provided herein. These methods enable the spatial detection of nucleic acids in situ within cell or tissue samples. These methods offer a significant advantage in assessing where and to what extent constructs are present, as they enable this with remarkable efficiency and accuracy. This is particularly important given that in-situ sequencing techniques are inefficient and have low detection efficiencies (see Moffitt JR, et al. Nat Rev Genet. 2022 Dec;23(12):741-759), and in particular, detection efficiencies have been reported to be 5-10 times lower in FFPE samples compared to fresh-frozen samples using other genomic techniques (see Moses L, et al. Nat Methods. 2022 May;19(5):534-546). Furthermore, RNA content varies significantly across cell types and tissues (see Walker DG, et al. Cell Tissue Bank. 2016 Sep;17(3):361-375), and RNA content affects gene detection rates by genome analysis techniques (see Mereu E, et al. Nat Biotechnol. 2020 Jun;38(6):747-755), thus necessitating new approaches for more efficient nucleic acid detection. Therefore, these methods enable screening of more constructs (each with its own unique barcode) with fewer sequencing iterations, improving both time and cost efficiency. These advantages could lead to faster screening, lead selection, or lead optimization of drug candidates, including gene therapies, cell therapies, or biologics. These advantages could also lead to a better understanding of the roles of different genes in biological systems. These are just some of the advantages and technical effects of the methods and compositions provided herein.

[0189] Embedding by reference All disclosures of each patent and scientific document referenced herein are incorporated by reference for all purposes.

[0190] Equivalents The present invention can be implemented in other specific forms without departing from its essence or main features. Accordingly, the embodiments described above should be construed as illustrative in all respects and not limit the invention as described herein. Accordingly, the scope of the invention is indicated not by the foregoing description but by the appended claims, and all modifications that fall within the meaning of the claims and their equivalents are intended to be incorporated into the invention.

Claims

1. A method for determining in situ the presence, quantity, and / or localization of a target nucleic acid sequence in one or more fixed mammalian cells within a biological sample, (a) In one or more fixed mammalian cells, (i) a DNA molecule containing a target nucleic acid sequence operably linked to a sequence-specific RNA polymerase promoter and (ii) a sequence-specific RNA polymerase are reacted to produce an RNA transcript of the target nucleic acid sequence. (b) Reacting the RNA transcript with reverse transcriptase in situ to produce a cDNA molecule containing the target nucleic acid sequence, and (c) Sequencing the cDNA molecule in situ to visualize the target nucleic acid sequence in one or more fixed mammalian cells. The method, including the method described above.

2. The method according to claim 1, further comprising, prior to step (a), contacting the immobilized mammalian cells with RNase to degrade endogenous RNA molecules.

3. The method according to claim 1 or 2, wherein the DNA molecule is an exogenous nucleic acid molecule introduced into one or more mammalian cells before fixation, or is derived from the exogenous nucleic acid molecule.

4. The method according to claim 3, wherein the target nucleic acid sequence is a barcode polynucleotide.

5. The method according to claim 3 or 4, wherein the exogenous nucleic acid molecule is incorporated into the genome of one or more mammalian cells by viral transduction, site-specific nuclease, or site-specific recombinase.

6. The method according to claim 5, wherein the exogenous DNA molecule is introduced into one or more mammalian cells using a viral vector selected from a lentiviral vector, retroviral vector, adenovirus vector, HSV vector, baculovirus vector, virus-like particle, pseudotype virus-like capsid, oncolytic virus vector, or AAV vector.

7. The method according to claim 5 or 6, wherein the exogenous nucleic acid sequence is incorporated at a specific site within the genome.

8. The method according to claim 5 or 6, wherein the exogenous nucleic acid sequence is incorporated at a random site within the genome.

9. The method according to claim 3 or 4, wherein the exogenous nucleic acid is not incorporated into the mammalian chromosome.

10. The method according to claim 9, wherein the exogenous nucleic acid molecule is retained in the nucleus of one or more mammalian cells.

11. The method according to claim 9 or 10, wherein the exogenous nucleic acid molecule is contained within a plasmid or artificial chromosome.

12. The method according to claim 1 or 2, wherein the target nucleic acid sequence is an endogenous nucleic acid sequence and the promoter is an exogenous promoter.

13. The method according to claim 12, wherein the endogenous nucleic acid sequence is variable among cells in the biological sample.

14. The method according to claim 13, wherein the endogenous nucleic acid sequence encodes a region including a T cell receptor, a B cell receptor, an immunoglobulin sequence, a repeat sequence, or a somatic mutation.

15. The method according to claim 12, wherein the target nucleic acid sequence is an endogenous sequence that does not change between cells in the biological sample.

16. The DNA molecule is generated in one or more fixed mammalian cells by reverse transcription using a DNA primer that hybridizes to a target RNA containing the desired nucleic acid sequence, and the DNA primer is (A) A 5' nucleic acid sequence containing a sequence-specific RNA polymerase promoter, and (B) A 3' nucleic acid sequence complementary to a portion of the target RNA adjacent to the target nucleic acid sequence. The method according to any one of claims 1 to 15, including the method described in any one of claims 1 to 15.

17. The method according to claim 16, further comprising converting the DNA molecule into double-stranded DNA by second-strand synthesis.

18. The method according to claim 16, wherein the 5' nucleic acid sequence of the DNA primer containing the sequence-specific RNA polymerase promoter is dsDNA.

19. The method according to claim 18, wherein the dsDNA is a hybridized dsDNA or a hairpin.

20. The method according to any one of claims 16 to 19, wherein the target RNA molecule is digested whole or partially following the synthesis of the DNA molecule.

21. The method according to any one of claims 1 to 20, wherein the in situ sequencing is sequencing by synthesis, sequencing by ligation, or sequencing by avidity.

22. The method according to claim 21, wherein the in situ sequencing is sequencing by synthesis.

23. The method according to any one of claims 1 to 22, wherein the sequence-specific RNA polymerase promoter is a phage promoter or a transcriptional activity variant thereof, and the sequence-specific RNA polymerase is a phage RNA polymerase.

24. The sequence-specific RNA polymerase promoter and the sequence-specific RNA polymerase are (i) Each of the following: T7 promoter, or its transcriptional activity variant, and T7 RNA polymerase, (ii) Each of the following: T3 promoter or its transcriptional activity variant, and T3 RNA polymerase, (iii) The SP6 promoter, or its transcriptional variant, and the SP6 RNA polymerase, respectively. A method according to any one of claims 1 to 23, selected from the group consisting of the following.

25. The method according to any one of claims 1 to 24, wherein the promoter is a T7 promoter and the RNA polymerase is a T7 RNA polymerase.

26. The method according to any one of claims 1 to 22, wherein the sequence-specific RNA polymerase promoter is a bacterial promoter and the sequence-specific RNA polymerase is a bacterial RNA polymerase.

27. The method according to any one of claims 1 to 22, wherein the sequence-specific RNA polymerase promoter is a eukaryotic promoter and the sequence-specific RNA polymerase is a eukaryotic RNA polymerase.

28. The method according to any one of claims 1 to 22, wherein the sequence-specific RNA polymerase promoter is a viral promoter and the sequence-specific RNA polymerase is a viral RNA polymerase.

29. The method according to any one of claims 1 to 22, wherein the sequence-specific RNA polymerase promoter is a synthetic promoter and the sequence-specific RNA polymerase is a synthetic RNA polymerase.

30. The method according to any one of claims 1 to 29, wherein the DNA molecule further comprises a transcription terminator.

31. The method according to claim 30, wherein the transfer terminator is a T7 terminator.

32. The method according to any one of claims 1 to 31, wherein the biological sample is immobilized using a solution containing formaldehyde and / or paraformaldehyde.

33. The method according to claim 32, wherein the solution contains 4% paraformaldehyde.

34. The method according to any one of claims 1 to 33, wherein the biological sample comprises a formalin-fixed paraffin-embedded (FFPE) sample containing one or more mammalian cells.

35. The method according to any one of claims 1 to 31, wherein the biological sample is fixed by freezing.

36. The method according to claim 35, wherein the sample comprises an optimal cleavage temperature compound, a hydrogel matrix, or a swellable polymer hydrogel.

37. The method according to any one of claims 1 to 31, wherein the sample is fixed using a solution containing alcohol.

38. The method according to claim 37, wherein the alcohol is methanol or ethanol.

39. The method according to any one of claims 1 to 31, wherein the sample is immobilized using a solution containing glutaraldehyde.

40. The method according to any one of claims 1 to 39, wherein the target nucleic acid sequence is less than 100 nucleotides long, less than 90 nucleotides long, less than 80 nucleotides long, less than 70 nucleotides long, less than 60 nucleotides long, less than 50 nucleotides long, less than 40 nucleotides long, less than 30 nucleotides long, less than 25 nucleotides long, less than 20 nucleotides long, less than 15 nucleotides long, less than 10 nucleotides long, or less than 5 nucleotides long.

41. The method according to any one of claims 1 to 40, wherein the DNA molecule further comprises one or more polynucleotide sequences encoding an exogenous protein, an endogenous protein, or a mixture of an exogenous protein and an endogenous protein.

42. The method according to any one of claims 1 to 41, wherein the DNA molecule further comprises a polynucleotide sequence encoding one or more exogenous proteins.

43. The method according to claim 42, wherein at least one subset of the one or more exogenous proteins is a synthetic protein and / or a chimeric protein.

44. The method according to claim 42 or 43, wherein the one or more exogenous proteins are independently selected from the group consisting of chimeric antigen receptors (CARs), antibodies, T cell receptors, cytokines, cell surface receptors, transcription factors, signaling proteins, and proteases.

45. The method according to any one of claims 42 to 44, wherein two or more exogenous proteins are expressed.

46. The method according to any one of claims 42 to 45, wherein the expression of the exogenous protein is controlled by an endogenous protein in one or more mammalian cells.

47. The method according to any one of claims 1 to 46, wherein the DNA molecule further comprises a polynucleotide sequence encoding an endogenous protein.

48. The method according to any one of claims 1 to 46, wherein the DNA molecule further comprises a polynucleotide sequence encoding endogenous RNA.

49. The method according to any one of claims 1 to 48, wherein the DNA molecule further comprises a polynucleotide sequence encoding exogenous RNA.

50. The method according to any one of claims 1 to 49, wherein the DNA molecule further comprises a polynucleotide sequence encoding nucleic acid sequences that modify the expression, function, and / or sequence of one or more genes.

51. The method according to claim 50, wherein the nucleic acid sequence that modifies the expression, function, and / or sequence of one or more genes is selected from the group consisting of sgRNA, gRNA, shRNA, and miRNA.

52. The method according to any one of claims 1 to 51, wherein the DNA molecule further comprises a polynucleotide sequence encoding a viral genome.

53. The method according to claim 52, wherein the viral genome is an oncolytic viral genome.

54. The method according to any one of claims 1 to 53, wherein the DNA molecule comprises a second sequence-specific RNA polymerase promoter configured to drive the transcription of a second nucleic acid sequence of interest in the presence of a second sequence-specific RNA polymerase.

55. The method according to claim 54, wherein the second nucleic acid sequence for the above purpose is a second barcode polynucleotide.

56. The second sequence-specific RNA polymerase promoter and the second sequence-specific RNA polymerase are (i) Each of the following: T7 promoter, or its transcriptional activity variant, and T7 RNA polymerase, (ii) Each of the following: T3 promoter or its transcriptional activity variant, and T3 RNA polymerase, (iii) The SP6 promoter, or its transcriptional variant, and the SP6 RNA polymerase, respectively. The method according to claim 54 or 55, selected from the group consisting of the following.

57. The method according to any one of claims 54 to 56, wherein the second sequence-specific RNA polymerase promoter is a T7 promoter or a transcriptional activity variant thereof, and the second sequence-specific RNA polymerase is a T7 RNA polymerase.

58. The method according to claim 54 or 55, wherein the second sequence-specific RNA polymerase promoter is a bacterial promoter or a transcriptionally active variant thereof, and the second sequence-specific RNA polymerase is a bacterial RNA polymerase.

59. The method according to claim 54 or 55, wherein the second sequence-specific RNA polymerase promoter is a eukaryotic promoter or a transcriptional activity variant thereof, and the second sequence-specific RNA polymerase is a eukaryotic RNA polymerase.

60. The method according to claim 54 or 55, wherein the second sequence-specific RNA polymerase promoter is a viral promoter or a transcriptional activity variant thereof, and the second sequence-specific RNA polymerase is a viral RNA polymerase.

61. The method according to claim 54 or 55, wherein the second sequence-specific RNA polymerase promoter is a synthetic promoter and the second sequence-specific RNA polymerase is a synthetic RNA polymerase.

62. The method according to any one of claims 54 to 61, wherein the first and second promoters and the RNA polymerase are the same.

63. The method according to any one of claims 54 to 61, wherein the first and second promoters and the RNA polymerase are different.

64. The method according to any one of claims 54 to 63, wherein the target nucleic acid sequence and the second target nucleic acid sequence are adjacent to a polynucleotide encoding the exogenous protein.

65. The method according to any one of claims 54 to 64, wherein the above-mentioned nucleic acid sequence and the above-mentioned second nucleic acid sequence are introduced onto the same nucleic acid.

66. The method according to any one of claims 54 to 64, wherein the above-mentioned nucleic acid sequence and the above-mentioned second nucleic acid sequence are introduced onto different nucleic acids.

67. The method according to any one of claims 54 to 66, wherein the DNA molecule comprises three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more sequence-specific RNA polymerase promoters, each configured to drive the transcription of a different nucleic acid sequence of interest in the presence of a specific sequence-specific RNA polymerase.

68. The method according to any one of claims 1 to 67, wherein the DNA molecule further comprises a first padlock binding sequence and a second padlock binding sequence, the first padlock binding sequence and the second padlock binding sequence being adjacent to a region containing the target nucleic acid sequence.

69. The method according to any one of claims 54 to 68, wherein the DNA molecule further comprises a third padlock binding sequence and a fourth padlock binding sequence, the third padlock binding sequence and the fourth padlock binding sequence being adjacent to a region comprising the second nucleic acid sequence of the objective.

70. Before in situ sequencing, step (c) is performed. (i) The step of contacting the cDNA with a first padlock probe including a 5' end and a 3' end, wherein the first padlock probe includes a 5' nucleic acid sequence inversely complementary to the first padlock binding site and a 3' nucleic acid sequence inversely complementary to the second padlock binding site, so that the 5' and 3' nucleic acid sequences can hybridize to the cDNA, (ii) The step of extending the 3' end of the first padlock probe via the target nucleic acid sequence using DNA polymerase, (iii) The step of generating a circular DNA template containing a nucleic acid sequence inversely complementary to the target nucleic acid sequence by ligating the 5' end of the padlock probe to the extended 3' end of the padlock probe, and (iv) The step of generating additional copies of the desired nucleic acid sequence using rolling circle amplification of the DNA template. The method according to claim 68 or 69, further comprising:

71. The method according to claim 70, wherein step (i) further comprises contacting the cDNA with a second padlock probe having a 5' end and a 3' end, the second padlock probe comprising a 5' nucleic acid sequence inversely complementary to the third padlock binding site and a 3' nucleic acid sequence inversely complementary to the fourth padlock binding site, and step (ii) further comprises extending the 3' end of the second padlock probe via the desired second nucleic acid sequence using a DNA polymerase.

72. The method according to any one of claims 69 to 71, wherein the first and second padlock binding sequences are different from the third and fourth padlock binding sequences.

73. The method according to any one of claims 69 to 71, wherein the first and second padlock binding sequences are identical to the third and fourth padlock binding sequences.

74. The method according to any one of claims 1 to 67, wherein the in situ sequencing is performed directly on the cDNA.

75. The method according to any one of claims 1 to 74, wherein the biological sample comprises one or more immune cells.

76. The method according to claim 75, wherein the one or more immune cells are T cells, NK cells, B cells, mast cells, dendritic cells, macrophages, neutrophils, basophils, and / or eosinophils.

77. The method according to any one of claims 1 to 76, wherein the biological sample comprises a mixture of cells derived from different species.

78. The method according to claim 77, wherein the biological sample includes human cells and mouse cells.

79. The method according to claim 77 or 78, wherein the biological sample comprises human immune cells and mouse cells.

80. The method according to any one of claims 1 to 76, wherein one or more cells in the biological sample are composed of cells derived from a single species.

81. The method according to claim 80, wherein one or more cells in the biological sample are composed of human cells.

82. The method according to any one of claims 1 to 81, wherein the biological sample comprises cancer cells and fibroblasts, or both.

83. The method according to any one of claims 1 to 82, wherein the biological sample comprises one or more human cancer cells.

84. The method according to any one of claims 1 to 83, wherein the biological sample comprises one or more mouse cancer cells.

85. The method according to any one of claims 1 to 84, wherein the biological sample comprises one or more nerve cells.

86. The method according to claim 85, wherein the nerve cells include one or more of neurons, astrocytes, and microglia.

87. The method according to any one of claims 1 to 86, wherein less than 100% of the cells in the biological sample contain the target nucleic acid sequence.

88. The method according to claim 87, wherein the biological sample comprises an FFPE sample, and less than 100% of the cells in the biological sample contain the target nucleic acid sequence.

89. The method according to any one of claims 1 to 86, wherein all or substantially all cells in the biological sample contain the nucleic acid sequence of the objective.

90. A method for determining in situ the presence, quantity, and / or localization of a target nucleic acid sequence in one or more fixed mammalian cells within a biological sample, (a) In one or more fixed mammalian cells, reverse transcription of a target RNA containing the target nucleic acid sequence using a DNA primer to generate a first cDNA molecule containing the target nucleic acid sequence, wherein the DNA primer is (i) A 5' nucleic acid sequence containing a sequence-specific RNA polymerase promoter, and (ii) A 3' nucleic acid sequence complementary to a portion of the target RNA adjacent to the target nucleic acid sequence. Includes, The DNA primer hybridizes to the target RNA, The first cDNA molecule comprises the sequence-specific RNA polymerase promoter operably linked to the target nucleic acid sequence, (b) Reacting the first cDNA molecule with a sequence-specific RNA polymerase to produce an RNA transcript containing the target nucleic acid sequence. (c) Reacting the RNA transcript with reverse transcriptase to produce a second cDNA molecule containing the target nucleic acid sequence, and (d) Sequencing the second cDNA molecule in situ to visualize the target nucleic acid sequence in one or more fixed mammalian cells. The method, including the method described above.

91. The method according to claim 90, further comprising converting the first cDNA molecule into double-stranded DNA using second-strand synthesis before step (b).

92. The method according to claim 90, wherein the 5' nucleic acid sequence of the DNA primer containing the sequence-specific RNA polymerase promoter is dsDNA.

93. The method according to claim 92, wherein the dsDNA is a hybridized dsDNA or a hairpin.

94. The method according to any one of claims 90 to 93, comprising, prior to step (b), contacting the fixed mammalian cells with RNase to degrade endogenous RNA molecules.

95. The method according to any one of claims 90 to 94, wherein the target nucleic acid sequence is an exogenous nucleic acid sequence introduced into one or more mammalian cells before fixation, or is derived from the exogenous nucleic acid sequence.

96. The method according to claim 95, wherein the target nucleic acid sequence is a barcode polynucleotide.

97. The method according to claim 95 or 96, wherein the exogenous nucleic acid sequence is incorporated into the genome of one or more mammalian cells by viral transduction, site-specific nuclease, or site-specific recombinase.

98. The method according to claim 97, wherein the exogenous nucleic acid sequence is introduced into the mammalian cell using a viral vector selected from a lentiviral vector, a retroviral vector, an adenovirus vector, an HSV vector, a baculovirus vector, a virus-like particle, a pseudotype virus-like capsid, or an AAV vector.

99. The method according to claim 97 or 98, wherein the exogenous nucleic acid sequence is incorporated at a pre-selected location within the genome.

100. The method according to claim 97 or 98, wherein the exogenous nucleic acid sequence is incorporated at a random position within the genome.

101. The method according to claim 95 or 96, wherein the exogenous nucleic acid is not incorporated into the mammalian chromosome.

102. The method according to claim 101, wherein the exogenous nucleic acid is retained in the nucleus of one or more cells.

103. The method according to claim 101 or 102, wherein the exogenous nucleic acid is contained within a plasmid or artificial chromosome.

104. The method according to any one of claims 90 to 94, wherein the target nucleic acid sequence is an endogenous sequence that changes between cells in the biological sample.

105. The method according to claim 104, wherein the endogenous nucleic acid sequence encodes a region including a T cell receptor, a B cell receptor, an immunoglobulin sequence, a repeat sequence, or a somatic mutation.

106. The method according to any one of claims 90 to 94, wherein the target nucleic acid sequence is an endogenous sequence that does not change between cells in the biological sample.

107. The method according to any one of claims 90 to 106, wherein the RNA molecule is mRNA.

108. The method according to any one of claims 90 to 106, wherein the RNA molecule is a non-coding RNA.

109. The method according to any one of claims 90 to 108, wherein the RNA molecule includes gRNA.

110. The method according to any one of claims 90 to 109, wherein the in situ sequencing is sequencing by synthesis, sequencing by ligation, or sequencing by avidity.

111. The method according to claim 110, wherein the in situ sequencing is sequencing by synthesis.

112. The method according to any one of claims 90 to 111, wherein the target nucleic acid sequence is less than 100 nucleotides long, less than 90 nucleotides long, less than 80 nucleotides long, less than 70 nucleotides long, less than 60 nucleotides long, less than 50 nucleotides long, less than 40 nucleotides long, less than 30 nucleotides long, less than 25 nucleotides long, less than 20 nucleotides long, less than 15 nucleotides long, less than 10 nucleotides long, or less than 5 nucleotides long.

113. The method according to any one of claims 90 to 112, wherein the promoter is a phage promoter or a transcriptional activity variant thereof, and the sequence-specific RNA polymerase is a phage RNA polymerase.

114. The promoter and the sequence-specific RNA polymerase (i) Each of the following: T7 promoter, or its transcriptional activity variant, and T7 RNA polymerase, (ii) Each of the following: T3 promoter or its transcriptional activity variant, and T3 RNA polymerase, (iii) The SP6 promoter, or its transcriptional variant, and the SP6 RNA polymerase, respectively. A method according to any one of claims 90 to 113, selected from the group consisting of the following.

115. The method according to any one of claims 90 to 114, wherein the promoter is a T7 promoter or a transcriptional activity variant thereof, and the RNA polymerase is a T7 RNA polymerase.

116. The method according to any one of claims 90 to 112, wherein the sequence-specific RNA polymerase promoter is a bacterial promoter or a transcriptional variant thereof, and the sequence-specific RNA polymerase is a bacterial RNA polymerase.

117. The method according to any one of claims 90 to 112, wherein the sequence-specific RNA polymerase promoter is a eukaryotic promoter or a transcriptional activity variant thereof, and the sequence-specific RNA polymerase is a eukaryotic RNA polymerase.

118. The method according to any one of claims 90 to 112, wherein the sequence-specific RNA polymerase promoter is a viral promoter or a transcriptional variant thereof, and the sequence-specific RNA polymerase is a viral RNA polymerase.

119. The method according to any one of claims 90 to 112, wherein the sequence-specific RNA polymerase promoter is a synthetic promoter and the sequence-specific RNA polymerase is a synthetic RNA polymerase.

120. The method according to any one of claims 90 to 119, wherein the biological sample is immobilized using a solution containing formaldehyde and / or paraformaldehyde.

121. The method according to claim 120, wherein the solution contains 4% paraformaldehyde.

122. The method according to any one of claims 90 to 121, wherein the biological sample comprises a formalin-fixed paraffin-embedded (FFPE) sample containing one or more mammalian cells.

123. The method according to any one of claims 90 to 119, wherein the biological sample is fixed by freezing.

124. The method according to claim 123, wherein the sample comprises an optimal cleavage temperature compound, a hydrogel matrix, or a swellable polymer hydrogel.

125. The method according to any one of claims 90 to 119, wherein the sample is fixed using a solution containing alcohol.

126. The method according to claim 125, wherein the alcohol is methanol or ethanol.

127. The method according to any one of claims 90 to 119, wherein the sample is immobilized using a solution containing glutaraldehyde.

128. The method according to any one of claims 90 to 127, wherein the target RNA encodes an exogenous protein, an endogenous protein, or a mixture of an exogenous protein and an endogenous protein.

129. The method according to any one of claims 90 to 128, wherein the target RNA encodes an exogenous protein.

130. The method according to claim 129, wherein the exogenous protein is a synthetic protein and / or a chimeric protein.

131. The method according to claim 130, wherein the exogenous protein is selected from the group consisting of chimeric antigen receptors (CARs), antibodies, T cell receptors, cytokines, cell surface receptors, transcription factors, signaling proteins, and proteases.

132. The method according to claim 130 or 131, wherein the expression of the exogenous protein is controlled by an endogenous protein in one or more mammalian cells.

133. The method according to any one of claims 90 to 127, wherein the target RNA encodes a nucleic acid sequence that alters the expression, function, and / or sequence of one or more genes, and the nucleic acid sequence is selected from the group consisting of sgRNA, gRNA, shRNA, and miRNA.

134. The method according to any one of claims 90 to 133, wherein the target RNA further comprises a first padlock binding sequence and a second padlock binding sequence, the first padlock binding sequence and the second padlock binding sequence being adjacent to the target nucleic acid sequence.

135. Before performing the in situ sequencing, step (d) (i) The step of contacting the second cDNA molecule with a padlock probe having a 5' end and a 3' end, wherein the padlock probe includes a 5' nucleic acid sequence inversely complementary to the first padlock binding site and a 3' nucleic acid sequence inversely complementary to the second padlock binding site, so that the 5' and 3' nucleic acid sequences of the padlock probe can hybridize to the cDNA, (ii) A step of extending the 3' end of the padlock probe using DNA polymerase, (iii) The step of generating a circular DNA template containing a nucleic acid sequence inversely complementary to the target nucleic acid sequence by ligating the 5' end of the padlock probe to the extended 3' end of the padlock probe, and (iv) The step of generating additional copies of the desired nucleic acid sequence using rolling circle amplification of the DNA template. The method according to claim 134, further comprising:

136. The method according to any one of claims 90 to 135, wherein the biological sample comprises one or more immune cells.

137. The method according to claim 136, wherein the one or more immune cells are T cells, NK cells, B cells, mast cells, dendritic cells, macrophages, neutrophils, basophils, and / or eosinophils.

138. The method according to any one of claims 90 to 137, wherein the biological sample comprises a mixture of cells derived from different species.

139. The method according to claim 138, wherein the biological sample includes human cells and mouse cells.

140. The method according to claim 138 or 139, wherein the biological sample comprises human immune cells and mouse cells.

141. The method according to any one of claims 90 to 137, wherein one or more cells in the biological sample are composed of cells derived from a single species.

142. The method according to claim 141, wherein one or more cells in the biological sample are composed of human cells.

143. The method according to any one of claims 90 to 142, wherein less than 100% of the cells in the biological sample contain the target nucleic acid sequence.

144. The method according to claim 143, wherein the biological sample comprises an FFPE sample, and less than 100% of the cells in the biological sample contain the target nucleic acid sequence.

145. The method according to any one of claims 90 to 142, wherein all or substantially all cells in the biological sample contain the nucleic acid sequence of the objective.

146. The method according to any one of claims 1 to 145, further comprising detecting the presence, quantity and / or localization of one or more additional analytes within the cell.

147. The method according to claim 146, wherein the one or more additional analytes are independently selected from the group consisting of proteins, RNA, DNA stained in a non-sequence-specific manner, DNA having a specific sequence, DNA mutations, lipids including but not limited to phospholipids and sphingolipids, carbohydrates including but not limited to monosaccharides and polysaccharides, metabolites, small molecules, cellular structures, and tissue structures.

148. The method according to any one of claims 1 to 147, further comprising detecting the presence, amount and / or localization of one or more protein analytes within the cell.

149. The method according to claim 148, wherein one or more protein analytes are detected by immunofluorescence microscopy.

150. The method according to any one of claims 1 to 149, further comprising detecting the presence, quantity and / or localization of one or more RNA analytes within the cell.

151. The method according to claim 150, wherein one or more RNA analytes are detected by a hybridization chain reaction (HCR).

152. (a) Sequence-specific RNA polymerase, (b) Reverse transcriptase, (c) Reagents for in situ sequencing, and (d) Instructions for using components (a) to (c) in situ to determine the presence, quantity, and / or localization of a target nucleic acid sequence in one or more fixed mammalian cells in a biological sample. A kit that includes this.

153. (i) The in situ sequencing is a synthetic sequencing, and the reagent for the in situ sequencing comprises (A) a plurality of detectably labeled nucleotides and (B) DNA polymerase, (ii) The in situ sequencing is ligation sequencing, and the reagent for the in situ sequencing comprises (A) a plurality of detectably labeled oligonucleotides containing a degenerate base and (B) a DNA ligase, or (iii) The in situ sequencing is avidity sequencing, and the reagent for the in situ sequencing comprises (A) a plurality of detectably labeled avidites and (B) a manipulated DNA polymerase, The kit according to claim 152.

154. (i) (A) A 3' nucleic acid sequence complementary to a portion of the target RNA adjacent to the target nucleic acid sequence, and (B) 5' nucleic acid sequence containing a sequence-specific RNA polymerase promoter DNA primers containing, or Further instructions for designing the aforementioned DNA primers, and (ii) Further instructions for reverse transcribing the target RNA using the DNA primers to generate a cDNA molecule The kit according to claim 152 or 153, further comprising:

155. The kit according to claim 154, wherein the 5' nucleic acid sequence of the DNA primer containing the sequence-specific RNA polymerase promoter is dsDNA.

156. The kit according to claim 155, wherein the dsDNA is hybridized dsDNA or a hairpin.

157. (i) a reagent for performing a second strand synthesis on the cDNA molecule, and / or (ii) further instructions for performing the second strand synthesis, according to claim 156.

158. A population of manipulated cells comprising an exogenous promoter juxtaposed with an endogenous genomic region containing a genomic sequence that is variable among cells in the population, wherein the exogenous promoter is capable of driving the expression of the genomic sequence that is variable among cells in the population, and the exogenous promoter is inserted in a site-specific manner.

159. The manipulated cell population according to claim 158, wherein the exogenous promoter is selected from the group consisting of the T7 promoter, the T3 promoter, and the SP6 promoter.

160. The manipulated cell population according to claim 158 or 159, wherein the promoter is a T7 promoter.

161. A population of manipulated cells according to any one of claims 158 to 160, wherein the exogenous promoter and the genomic sequence which is variable among cells in the population are separated at approximately 2 kilobases (kb) or less, 1.5 kb or less, 1 kb or less, 900 (base pairs) bp or less, 800 bp or less, 700 bp or less, 600 bp or less, 500 bp or less, 400 bp or less, 300 bp or less, 250 bp or less, 200 bp or less, 150 bp or less, 100 bp or less, 50 bp or less.

162. The manipulated population of cells according to claim 156, wherein the separation is located within the genomic DNA sequence, exon coding sequence, or RNA sequence of the cells.

163. The population of manipulated cells according to any one of claims 158 to 162, wherein the manipulated cells are mammalian cells.

164. The manipulated cell population according to claim 163, wherein the mammalian cells are human cells.

165. The population of manipulated cells according to claim 163 or 164, wherein the manipulated cells are immune cells.

166. The manipulated cell population according to claim 165, wherein the immune cells are T cells, NK cells, B cells, mast cells, dendritic cells, macrophages, neutrophils, basophils, and / or eosinophils.

167. The manipulated population of cells according to any one of claims 158 to 166, wherein the genome sequence, which is variable among cells within the population, encodes a region including a T cell receptor, a B cell receptor, an immunoglobulin sequence, a repetitive sequence, or a somatic mutation.

168. A method for determining in situ the presence, quantity, and / or localization of a target nucleic acid sequence in one or more mammalian cells within a biological sample, (a) Introducing an exogenous DNA molecule containing the target nucleic acid sequence operably linked to a sequence-specific RNA polymerase promoter into one or more mammalian cells. (b) Fixing the mammalian cells, (c) Reacting the exogenous DNA molecule with a sequence-specific RNA polymerase to produce an RNA transcript of the target nucleic acid sequence. (d) Reacting the RNA transcript with reverse transcriptase in situ to produce a cDNA molecule containing the target nucleic acid sequence, and (e) Sequencing the cDNA molecule in situ to visualize copies of the target nucleic acid sequence in one or more fixed mammalian cells. The method, including the method described above.

169. The method according to claim 168, wherein the exogenous DNA molecule is genetically engineered within the genome of one or more mammalian cells.

170. The method according to claim 168 or 169, wherein the exogenous DNA molecule is genetically engineered upstream of one or more target gene loci.

171. A method for determining in situ the presence, quantity, and / or localization of a target nucleic acid sequence in one or more mammalian cells within a biological sample, (a) Fixing the mammalian cells, (b) In one or more mammalian cells, (i) A 5' nucleic acid sequence containing a sequence-specific RNA polymerase promoter, and (ii) A 3' nucleic acid sequence complementary to a portion of the target RNA adjacent to the target nucleic acid sequence. This involves introducing a DNA primer containing the following: The DNA primer hybridizes to the target RNA containing the desired nucleic acid sequence, and the introduction of the DNA primer hybridizes to the target RNA containing the desired nucleic acid sequence. (c) Reverse transcription of the target RNA using the DNA primer to generate a first cDNA molecule containing the target nucleic acid sequence operably linked to the target nucleic acid sequence. (d) Reacting the first cDNA molecule with a sequence-specific RNA polymerase to produce an RNA transcript containing the target nucleic acid sequence. (e) Reacting the RNA transcript with reverse transcriptase to produce a second cDNA molecule containing the target nucleic acid sequence, and (f) Sequencing the second cDNA molecule in situ to visualize the target nucleic acid sequence in one or more fixed mammalian cells. The method, including the method described above.

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