Imaging-based hybrid CRISPR screening

Abstract

The present invention relates to imaging cells for determining phenotypes and / or genotypes of cell populations or constructing genotype-phenotype correspondences for high throughput screening. The cells may be manipulated using CRISPR or other techniques. Nucleic acids may be introduced into cells using lentiviruses. The nucleic acid may contain a guide portion comprising a DNA or RNA recognition sequence, a reporter gene portion, and an identification portion comprising one or more read sequences. The guide moieties can be used to alter the phenotype of the cell, for example, using sequences that can be targeted with CRISPR or other techniques, such as sgRNA sequences, and the phenotype of the cell can be determined using various imaging approaches. The identification portion may be determined using MERFISH or other suitable techniques. In addition, correlation or co-localization between the determination of the reporter gene and the read sequence can greatly improve decoding accuracy. Other aspects relate to compositions or devices, kits, and the like for use in the methods.

Description

[0001] This application is a divisional application. The corresponding parent application has application number 202080043174.X, filing date April 17, 2020, and the name of the invention is “Imaging-based hybrid CRISPR screening”.

[0002] Related Applications

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 836,578, entitled “Imaging-Based Pooled CRISPR Screening,” filed by Zhuang et al. on April 19, 2019, and U.S. Provisional Patent Application Serial No. 62 / 841,715, entitled “Imaging-Based Pooled CRISPR Screening,” filed by Zhuang et al. on May 1, 2019. The entire contents of each of these applications are incorporated herein by reference.

[0004] Government funding

[0005] This invention was made with government support under Grant No. MH113094 awarded by the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0006] The present invention generally relates to imaging cells, for example, for determining the phenotype and / or genotype of a cell population. In some cases, cells can be manipulated, for example, using CRISPR or other techniques. Background Art

[0007] The development of CRISPR-based gene editing systems has greatly improved our ability to manipulate genes and explore the basic molecular mechanisms of cellular function through genetic perturbations. Facilitated by the ability to generate high-diversity nucleic acid libraries, CRISPR-based mixed library screening can greatly accelerate the discovery of genes involved in cellular processes. However, the phenotypes available in mixed library screening are essentially limited to cell viability and marker expression. In recent years, single-cell RNA sequencing and mass cytometry flow cytometry have been combined with CRISPR screening to expand the phenotypic space available for mixed library screening, allowing genetic screening based on single-cell RNA and protein expression profiles.

[0008] However, high-throughput pooled library screening still fails to exploit many important cellular phenotypes. These include the morphology of cellular structures and the intracellular molecular organization, as well as their dynamics, which can only be measured by techniques such as high-resolution imaging. High-content imaging further allows these properties to be measured simultaneously for many molecular species in a parallel mode - for example, recent advances in single-cell transcriptome imaging have increased the number of molecular phenotypes that can be imaged down to the genome scale in a single cell in a single experiment. Despite the ability of imaging to obtain cellular phenotypes, imaging-based pooled library screening remains challenging, primarily due to the difficulty in determining the genotype of individual cells whose phenotypes have been imaged in pooled library screening. Several approaches have been developed to determine genotypes by sequencing after physically isolating cells with a specific phenotype. However, in order to determine the full genotype-phenotype correspondence, full imaging-based pooled library screening approaches are needed, in which the genotype and phenotype of individual cells are imaged in situ. Summary of the invention

[0009] The present invention generally relates to imaging cells, e.g., for determining the phenotype and / or genotype of a cell population. In some cases, cells can be manipulated, e.g., using CRISPR or other techniques. The subject matter of the present disclosure in some cases relates to interrelated products, alternative solutions to specific technical problems, and / or a variety of different uses of one or more systems and / or articles.

[0010] In one aspect, the present invention generally relates to a method. According to one set of embodiments, the method comprises: (a) introducing DNA into a plurality of cells, the DNA comprising: a guide portion comprising a recognition sequence, a reporter gene portion, and an identification portion comprising a reading sequence; (b) determining the position of the RNA molecule expressed by the reporter gene portion of the introduced DNA in the plurality of cells by determining the reporter gene portion; (c) determining the reading sequence on the RNA molecule expressed by the introduced DNA comprising the reporter gene portion and the identification portion in the plurality of cells by exposing the cells to a readout probe capable of binding to the reading sequence; (d) co-localizing the binding of the readout probe with the position of the RNA molecule expressed by the reporter gene portion of the introduced DNA; (e) repeating (b), (c) and (d) multiple times using different reading sequences; and (f) forming a codeword corresponding to the binding of the co-localized readout probe, wherein the bit value of the codeword is based on the binding of the readout probe to the reading sequence.

[0011] According to another set of embodiments, the method includes: introducing DNA into multiple cells, the DNA comprising: a guide portion comprising a recognition sequence, a reporter gene portion, and an identification portion comprising a reading sequence; determining the positions of RNA molecules expressed by the reporter gene portion of the introduced DNA in the multiple cells by determining the reporter gene portion; determining the reading sequence in the multiple cells by exposing the cells to multiple readout probes, each of which can bind to the reading sequence; co-localizing the binding of the readout probes with the positions of the RNA molecules expressed by the reporter gene portion of the introduced DNA; and forming a codeword corresponding to the binding of the co-localized readout probes, wherein the bit value of the codeword is based on the binding of the readout probes to the reading sequence.

[0012] In another group of embodiments, the method includes: introducing a nucleic acid into a plurality of cells, wherein the nucleic acid comprises: a guide portion comprising a recognition sequence, a reporter gene portion, and an identification portion comprising a reading sequence; imaging the plurality of cells, wherein the cells exhibit imageable phenotypic differences due to expression of the guide portion; and acquiring a plurality of images of the plurality of cells, wherein the cell images exhibit differences due to differences in the identification portion of the nucleic acid within the cells.

[0013] In another group of embodiments, the method includes: introducing DNA into multiple cells using a lentivirus, wherein the DNA includes: a guide portion comprising a recognition sequence, a reporter gene portion, and an identification portion comprising a reading sequence; determining the phenotype of the multiple cells, and determining the genotype of the multiple cells, and determining the correspondence between the genotype and the phenotype.

[0014] According to yet another set of embodiments, the method includes: introducing DNA into multiple cells using a lentivirus, wherein the DNA includes: a guide portion including a recognition sequence and an identification portion including a reading sequence; determining the phenotypes of the multiple cells, determining the genotypes of the multiple cells, and determining the correspondence between the genotypes and the phenotypes.

[0015] In another aspect, the present invention encompasses methods of making one or more of the embodiments described herein. In yet another aspect, the present invention encompasses methods of using one or more of the embodiments described herein.

[0016] Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention considered in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying drawings, which are schematic and are not intended to be drawn to scale. In the accompanying drawings, each identical or nearly identical component illustrated is generally represented by a single numeral. For the sake of clarity, it is not necessary for each component to be labeled in each figure, nor is it necessary for each component of each embodiment of the invention shown to be labeled, wherein illustration is not necessary to allow one of ordinary skill in the art to understand the invention. In the drawings:

[0018] Figures 1A-1F Schematic diagram of imaging-based barcode detection for genotype determination according to one embodiment of the present invention;

[0019] Figures 2A-2D FIGURE 1 illustrates a bar code misidentification rate in another embodiment of the present invention;

[0020] Figures 3A-3E Schematic diagram of a lentiviral design in another embodiment of the present invention;

[0021] Figures 4A-4D Schematic diagram of imaging-based hybrid CRISPR screening in another embodiment of the present invention;

[0022] Figures 5A-5C The genetic factors involved in regulation are schematically illustrated according to one embodiment of the present invention;

[0023] Figure 6A-6B According to another embodiment of the present invention, certain genes for inhibiting transcription are illustrated;

[0024] Figure 7 Schematic diagram of a cloning strategy for a library in one embodiment of the present invention;

[0025] Figure 8 Schematic diagram of colocalization ratio analysis in another embodiment of the present invention;

[0026] Fig. 9 Schematic diagram of a cloning strategy for a library in another embodiment of the present invention;

[0027] Figures 10A-10D illustrates the knockdown of certain genes in one embodiment of the present invention; and

[0028] Fig.11 The diagram shows changes in MALAT1 nuclear speckle enrichment in another embodiment of the invention. DETAILED DESCRIPTION

[0029] The present invention generally relates to imaging cells, for example, for determining the phenotype and / or genotype of a cell population, for example, to construct a genotype-phenotype correspondence for high-throughput screening. In some cases, the cells can be manipulated, for example, using CRISPR or other techniques. In certain embodiments, a nucleic acid can be introduced into the cell, for example, using a lentivirus. The nucleic acid can include: a guide portion comprising a DNA or RNA recognition sequence, a reporter gene portion, and an identification portion comprising one or more read sequences. The guide portion can be used to change the phenotype of the cell, for example, using a sequence that can be targeted using CRISPR or other techniques, such as an sgRNA sequence, and in some cases, the phenotype of the cell can be determined using various imaging methods. The identification portion can be determined using MERFISH or other suitable techniques. In addition, in some cases, the association or co-localization between the determination of the reporter gene and the read sequence can greatly improve the decoding accuracy, for example, due to the misidentification of the reduced background signal. Other aspects generally relate to compositions or devices for use in such methods, kits for use in such methods, or the like.

[0030] An example aspect of the present invention generally relates to systems and methods for manipulating the genetic material of a cell (e.g., using CRISPR or other technologies) and determining the phenotype of the resulting cell as a result of the manipulation. The genotype of the cell can also be determined, for example, using a codeword encoding a read sequence, such as used in MERFISH or similar technologies. By determining how the genotype of the cell is changed and the phenotype of the cell is changed, certain embodiments as discussed herein can be used to understand complex problems, such as about cell morphology, subcellular molecular organization, etc., for example, the spatial structure within a cell (such as a mammalian cell).

[0031] Now refer to Figure 1A Describe an exemplary embodiment of the invention. In this figure, a nucleic acid library member can be introduced into a cell, such as a mammalian cell. In one set of embodiments, the nucleic acid comprises a guide portion (e.g., comprising an sgRNA or another recognition sequence that can be used to identify a target site), a reporter gene portion (e.g., can directly or indirectly generate a signal such as a fluorescence or immunoprecipitation signal), and an identifier or "barcode" portion (e.g., comprising a reading sequence that can be used to distinguish various nucleic acids that contain different guide portions from each other).

[0032] Various methods can be used to introduce nucleic acid into cells. These methods include, for example, viral delivery (e.g., using slow virus, retrovirus, adenovirus, adeno-associated virus, etc.), electroporation, bombardment delivery, etc. In some cases, slow virus can be useful because it allows nucleic acid to be stably integrated into the genome of the cell. In addition, in certain embodiments, the rate at which nucleic acid is introduced into the cell can be controlled so that most cells contain only one such nucleic acid. For example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the cells can have only one such nucleic acid introduced therein.

[0033] In this example, for the lentivirus generated by the mixed library, each lentivirus can include two library members. During the lentiviral infection process, the guide portion and the identification portion can be recombined. Such a recombination can lead to an incorrect identification of the guide portion based on the measurement of the identification portion. Therefore, in one set of embodiments, the guide portion and the identification portion can be placed in close proximity to each other in the 3'LTR region of the lentivirus, that is, after the polypurine band (PPT) sequence, so that the distance between the guide portion and the identification portion is minimized, for example, for the constant region of the sgRNA of Cas9, it is 100 bases or less. In this way, the recombination rate can be reduced to improve the accurate association between the guide portion and the identification portion. In some cases, the guide portion is replicated in the 5' region of the proviral DNA (e.g., lentivirus). This can cause the guide portion to be integrated into the host cell genome to provide expression of the guide portion.

[0034] After introduction, the cell can be studied to determine the phenotype of the cell and the genotype of the cell (e.g., using an identification moiety). For example, the phenotype can be measured using an imaging method that detects proteins, RNA, or DNA in a cell or a subcompartment of a cell, etc. In certain embodiments, the phenotype can also be associated with cell growth, morphology, or cell-cell interactions. In some cases, the phenotype can be a temporal change, a kinetic of a cell property, etc. In some cases, the phenotype can include multiple features, i.e., a multidimensional readout.

[0035] The identification portion can be determined, for example, using MERFISH (multiple error-corrected fluorescence in situ hybridization) or other techniques. Those of ordinary skill in the art are familiar with MERFISH and related techniques; see, for example, International Patent Application Publication Nos. WO2016 / 018960, WO 2016 / 018963, WO 2018 / 089445, WO 2018 / 218150, WO 2018 / 089438. In some cases, in some consecutive embodiments, the identification portion may include various "reading sequences" or nucleic acid sequences that can be specifically identified using corresponding nucleic acid probes (e.g., "reading probes"). In techniques such as MERFISH, the presence or absence of a read sequence can be encoded as a digit number (digit), and the sequence of the read probe can therefore be encoded as a codeword (codeword). In addition, in some cases, various error detection and / or correction techniques, such as Hamming codes or Golay codes, can be applied to codewords.

[0036] In some cases, the determination of the reporter gene portion can be performed at intervals with the determination of the various portions of the identification portion (e.g., using one or more read probes). In addition, in some embodiments, the association or co-location between the position of the reporter gene portion and the determination of the identification portion can be used to significantly improve decoding accuracy. For example, binding events or codewords that do not fully correspond to the position of the reporter gene portion can be ignored as background noise, non-specific markers, etc. This association or co-location between the reporter gene portion and the identification portion can significantly improve detection accuracy.

[0037] The above discussion is a non-limiting example of one embodiment of the invention that can be used to image cells (e.g., to determine the phenotype and / or genotype of a cell population). However, other embodiments are also possible. Therefore, more generally, various aspects of the invention relate to various systems and methods for determining the phenotype and / or genotype of a cell population (e.g., by imaging) and / or manipulating the cells using CRISPR or other techniques.

[0038] According to one aspect, the present invention generally relates to systems and methods for determining the phenotype and / or genotype of a cell population using imaging. In addition, the genome of the cell can, for example, be operated using CRISPR or other techniques. In some cases, a relatively large number of cells can, for example, be studied using suitable imaging techniques (such as those described herein) to determine their phenotype and genotype, for example, determined after operation. In some embodiments, due to the use of such imaging techniques, a relatively large number of cells can be determined, allowing for relatively large-scale or high-throughput screening, as discussed herein. For example, a specific phenotype of multiple cells can be determined (for example, after editing by CRISPR), and cells with a certain or desired phenotype can also be determined by genotype.

[0039] In some cases, a relatively large number of cells can be determined. For example, depending on the magnification, a single field of view can include a relatively large number of cells (e.g., at least 10, at least 100, at least 1,000, at least 10,000, at least 100,000 cells, etc.). In addition, the sample can be larger than a single field of view (e.g., especially under a relatively large magnification), and multiple images of different parts of the sample can be obtained manually or automatically (e.g., using computer control). This can allow for the study of even larger amounts of cells by using more than one field of view. For example, at least 10, at least 100, at least 1,000, at least 10,000, at least 100,000, at least 1,000,000, at least 10,000,000 cells, etc. For example, an overall image of a sample can be combined using multiple fields of view (e.g., fields of view taken simultaneously or nearly simultaneously) to produce an image; for example, at least 2, at least 3, at least 5, at least 7, at least 10, at least 15, at least 20, at least 30, at least 50, at least 75, or at least 100 images (e.g., corresponding to different portions of the sample) can be acquired at different fields of view to produce an overall image. Thus, in some cases, the sample can be substantially larger than a single field of view. For example, a sample can have at least about 0.01 cm 2 , at least about 0.03cm 2 , at least about 0.1cm 2 , at least about 0.3cm 2 , at least about 1cm 2 , at least about 3cm 2 , or at least about 10cm 2 Equal area.

[0040] In addition, in some embodiments, multiple images can be taken for the same field of view. For example, at least 2, at least 3, at least 5, at least 7, at least 10, at least 15, at least 20, at least 30, at least 50, at least 75, or at least 100 images can be acquired for the same field of view.

[0041] In some cases, in one set of embodiments, multiple images can be taken at each field of view imaged within the sample. In some embodiments, different wavelengths can be used. For example, in some cases, for example, different illumination sources can be used to collect images, and different filters can be used to capture the images to produce images of different colors that detect the presence of different fluorescent compounds. Thus, in some embodiments, different images can be taken at different wavelengths, for example, for viewing images in different colors (e.g., red-green-blue, red-yellow-blue, cyan-magenta-yellow, etc.).

[0042] In some embodiments, these images can be collected at limited time intervals to form a time-lapse image of the sample. This can be useful, for example, to determine time-varying characteristics, for example, the growth of cells. For example, an image (or multiple images) can be acquired at different time points, for example, using a cycle of about 5 seconds, about 10 seconds, about 15 seconds, about 30 seconds, about 1 minute, about 2 minutes, about 3 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 1 day, etc. Similarly, in some embodiments, images can be collected after different treatments are performed on the same sample.

[0043] In addition, in some embodiments, multiple images can be collected using different imaging modalities, such as super-resolution optical microscopy, conventional epifluorescence microscopy, confocal microscopy, etc., including those described herein. In some cases, these images can be merged to form high-content optical measurements of cell properties.

[0044] The cell can be any suitable cell, for example, a mammalian cell (e.g., a human or non-human cell), a bacterial cell (e.g., E. coli), a eukaryotic cell, a prokaryotic cell, a yeast cell, or a cell of other types. The cell can be from any suitable source, for example, a cell culture. In some cases, the cell can be taken from a tissue sample, for example, taken from a biopsy, artificially grown or cultured, or the like. In some cases, the cell is genetically modified. In some cases, a tissue sample can be analyzed. In certain embodiments, a plurality of cells can be transfected as discussed herein, and the resulting phenotype of the cell is determined.

[0045] In some aspects, nucleic acid is introduced into a cell, which can be used to modify the genetic material of the cell, e.g., its genome. Technologies such as CRISPR or other related technologies can be used to modify the genetic material of the cell, e.g., as guided by the nucleic acid. In some embodiments, this can allow accurate genetic manipulation of the identified cells, and their corresponding phenotypes, using the identified moieties to identify the genotypes that lead to the observed phenotypes.

[0046] For example, in one group of embodiments, the nucleic acid delivered to the cell may include a guide portion, and / or a reporter gene portion, and / or an identification portion. The guide portion may include an sgRNA or another recognition sequence that can be used to identify a target site (e.g., within the genome of the cell). The reporter gene portion may be capable of directly or indirectly generating a signal, such as a fluorescent signal. For example, the reporter gene portion may encode a fluorescent protein (e.g., GFP), an enzyme that can be used to make another molecule fluorescent (e.g., luciferase), an enzyme that produces a detectable chemical reactant, and the like. The identification portion may include sequences that can be used to distinguish various nucleic acids containing different guide portions from each other. For example, the identification portion may include one or more sequences (e.g., "reading sequences") that can be read using corresponding nucleic acid probes (e.g., "reading probes").

[0047] Guide part and / or reporter gene part and / or identification part, if present, can be arranged in any suitable order on the nucleic acid to be introduced into the cell. In some cases, these parts can be relatively close to each other (for example, in the nucleic acid, for example, less than 5,000, less than 3,000, less than 1,000, less than 500, less than 300, less than 100, less than 50, less than 30 or less than the distance of 10 bases). In addition, in some cases, for example, in the nucleic acid, one or more of these parts can overlap at least in part. In addition, in some embodiments, other parts or sequences may also be present in the nucleic acid. For example, one or more of these parts can include promoter sequences, such as those discussed herein.

[0048] In one set of embodiments, the nucleic acid includes an expression portion or a guide portion. The guide portion may include any suitable nucleic acid sequence that is suspected of being able to change the phenotype of the cell, and / or can be used to intentionally change or manipulate the genome of the cell, for example, resulting in an observable phenotypic change in the cell. For example, the guide portion may encode a gene, a protein, a regulatory sequence (e.g., an operator, a promoter such as a CMV promoter, a repressor, a transcription factor binding site, etc.), a sequence encoding a non-coding RNA (e.g., miRNA, siRNA, rRNA, tRNA, lncRNA, snoRNA, snRNA, exRNA, piRNA, tsRNA, rsRNA, shRNA, Cas9 guide RNA, sgRNA, etc.), and the like. In some cases, the guide portion may be part of the same nucleic acid that includes the identification portion; however, in other cases, the expression portion may be part of a different nucleic acid.

[0049] Thus, for example, the guide portion may include a sequence that recognizes a target region of interest (e.g., a target region of interest on DNA (e.g., on the genome of the cell)), such as an RNA sequence. In some cases, the guide portion may also include a binding sequence, such as a Cas binding sequence that can be recognized by Cas or another nuclease. For example, in some cases, the guide portion may be suitable for allowing CRISPR editing of a genome. For example, the guide portion may include a gRNA (guide RNA) or an sgRNA (single guide RNA). In some embodiments, the sgRNA may include a crispr RNA portion (crRNA), the crRNA portion being a sequence complementary to a target sequence (e.g., complementary to a target DNA), and a tracrRNA portion that can be recognized by a Cas nuclease or another nuclease. In some cases, the crRNA portion may have 17, 18, 19, or 20 nucleotides. A wide variety of different Cas nucleases can be used, such as Cas9 (from Streptococcus pyogenes), Cas14, CasX, CasY, Cas12a, Cas13a, Cas13b, Cas13d, Cas14a, etc. Variant forms of these Cas nucleases are also contemplated, for example, high-fidelity Cas9, eSpCas9, SpCas9-HF1, HypaCas9, FokI-fused dCas9, xCas9, dCas9, etc. Non-limiting examples of suitable binding sequences for Cas are provided below. In addition, one of ordinary skill in the art will be aware of CRISPR and related technologies, and kits that can be used to perform CRISPR experiments are readily commercially available.

[0050] In some embodiments, there may be more than one possibility for a guide portion. For example, a nucleic acid library may be prepared, e.g., with different crRNA portions, e.g., for binding to different target sequences in a genome. In some cases, there are at least 10, at least 10, or more possibilities for a guide portion. 2 Types, at least 10 3 Types, at least 10 4 Types, at least 10 5 The number of possible identification moieties may be different, for example, being able to bind to different target sites within the genome, and / or being able to cause different changes or manipulations of the genome, etc. Thus, in certain embodiments, one or more identification moieties (such as those described herein) and one or more guide moieties may be used to prepare a plurality of distinguishable nucleic acids. However, it should be understood that the number of possible identification moieties need not equal the number of possible guide moieties, i.e., some redundancy may be added, for example, as discussed below.

[0051] In some embodiments, the nucleic acid can include a reporter gene portion, which can be, for example, measured using fluorescence or other detection techniques. For example, the reporter gene portion can include a gene encoding a fluorescent protein, such as GFP (green fluorescent protein), a red fluorescent protein from dsRed, PAGFP, PSCFP, PSCFP2, Dendra, Dendra2, EosFP, tdEos, mEos2, mEos3, PAmCherry, PAtagRFP, mMaple, mMaple2 and mMaple3. Other suitable fluorescent proteins are known to those of ordinary skill in the art. See, for example, U.S. Patent No. 7,838,302 or U.S. Patent Application Serial No. 61 / 979,436, the entire contents of each of which are incorporated herein by reference.

[0052] In another set of embodiments, the reporter gene portion can encode an enzyme (e.g., luciferase) that can make another molecule become fluorescent. When expressed in a cell, a suitable substrate (e.g., luciferin) can be added, which can be converted into a fluorescent form when exposed to the enzyme. However, in areas where the nucleic acid is not present, no fluorescence occurs. In this way, the nucleic acid can be located or positioned in a cell (or in a part of the cell).

[0053] It should be understood that the reporter gene portion need not be determined only by fluorescence. Other reporter gene portions can be used in other embodiments. For example, in one embodiment, an enzyme that produces a detectable chemical reactant or analog can be encoded within the reporter gene portion. Other examples of reporter genes that can be used include, but are not limited to, proteins that can be detected by immunoprecipitation, immunofluorescence, etc. Non-limiting examples of suitable proteins include Myc tags or HA tags.

[0054] Any suitable technique can be used to determine the reporter gene portion, and the exact method can depend on the type of reporter gene. Examples include, but are not limited to, in situ hybridization techniques such as smFISH (single molecule fluorescence in situ hybridization), multiplex FISH, CASFISH, or other techniques known to those of ordinary skill in the art. In one embodiment, smFISH is used to locate the reporter gene portion, for example, in a cell.

[0055] In addition, as discussed herein, the position of the marker portion can also be determined and associated or co-localized with the reporter portion, which can, for example, be used to reduce background noise and / or improve decoding accuracy. For example, the reporter portion of the nucleic acid can produce a first signal (e.g., a first fluorescence), and the marker portion can produce a second signal (e.g., a second fluorescence, which can be at the same or different wavelength as the first fluorescence), and the two can be associated or co-localized with each other.

[0056] In some embodiments, the nucleic acid may include an identification portion or a "barcode" of nucleotides that can be used to distinguish multiple nucleic acids from each other. The identification portion may be present at any suitable position on the nucleic acid. For example, in one embodiment, the identification portion may be present in the 3'UTR of the reporter gene.

[0057] In some cases, other sequences may be present in the identification portion. For example, in some cases, the identification portion may include a promoter or another regulatory sequence (e.g., an operator, a promoter such as a CMV promoter, a repressor, a transcription factor binding site, etc.). A promoter may drive transcription. In some embodiments, the promoter of the identification portion may be the same or different from the promoter of the guide portion.

[0058] In certain embodiments, a library of marker moieties may be used, e.g., containing at least 10, at least 10 2 At least 10 3 At least 10 4 At least 10 5 At least 10 6 At least 10 7 At least 10 8The single sequence can be determined individually (e.g., randomly), although in some cases, the identification portion can be limited to multiple variable portions (or "bits"), for example, in order. For example, the identification portion can include at least 2, at least 3, at least 5, at least 7, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, or at least 50 variable portions. Each variable portion can include at least 2, at least 3, at least 4, at least 5, at least 7, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, or more possibilities.

[0059] Thus, for example, defining an identifier having 22 variable regions, each with 2 unique possibilities, would define an identifier having 2 22 =4,194,304 members of the identification portion library. As another non-limiting example, the identification portion can be defined as having 10 variable regions and each variable region has 7 unique possibilities, thereby defining a library with 7 10 The identification part library of each member. It should be understood that the variable part can include any suitable number of nucleotides, and the different variable parts in the identification part can independently have the same or different number of nucleotides. Different variable regions can also have the same or different unique possibilities.

[0060] For example, the variable portion can be limited to a length of at least 2, at least 3, at least 4, at least 5, at least 7, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, or more nucleotides, and / or a maximum length of no more than 50, no more than 40, no more than 30, no more than 25, no more than 20, no more than 15, no more than 10, no more than 7, no more than 5, no more than 4, no more than 3, or no more than 2 nucleotides. Combinations of these are also possible, for example, the variable portion can have a length of 5-50nt or 15-25nt, etc.

[0061] Each read sequence position can be considered to be a "bit" (e.g., 1 or 0 in the present example), although it should be understood that the number of possibilities for each "bit" is not necessarily limited to just 2, unlike in a computer. In other embodiments, there may be 3 possibilities (i.e., "trits"), 4 possibilities (i.e., "quad-bits"), 5 possibilities, etc., instead of just 2 possibilities. For example, various trits are used in the examples below. However, in some but not all embodiments, the use of bits (of any number of possibilities) to form the identification portion can allow the use of codewords, error detection codes, error correction codes, etc. within the identification portion, for example, as discussed in detail herein.

[0062] In some cases, the variable part of the identification part can be linked together to produce the identification part. However, in other cases, one or more variable parts can be separated, for example, with the constant part of nucleotides, thereby producing the identification part. In addition, in some cases, some or all possible variable parts in the library can be unique, for example, to reduce confusion as far as possible. Any method can be used for linkage. For example, a plurality of parts can be used to connect, overlap PCR, oligonucleotide pool synthesis or other techniques known to those of ordinary skill in the art for nucleic acid to be connected or linked together to link together.

[0063] In certain embodiments, all members of a library are generated and / or used. However, in other embodiments, all members of a library may not necessarily be generated and / or used. For example, in some embodiments, for example, to reduce or eliminate unclear or unintentional reuse, a smaller subset of the library may be used, for example, less than 75%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, or less than 0.1% of all possible members of the library are generated and / or used.

[0064] In some embodiments, the genotype of the cell can be determined, for example, using the identification portion to determine. A variety of different techniques for determining the genotype of a cell can be used, for example, FISH, smFISH, MERFISH, in situ hybridization, multiple FISH, CASFISH or other techniques known to those of ordinary skill in the art. In some embodiments, these methods may be related to direct hybridization with the identification portion, or to direct hybridization with a molecule generated by the portion by the cell. In some cases, it may also be related to the combination of separate adapter entities, which are then directly combined with the identification portion or the molecule generated by the identification portion. Other non-limiting examples of technology include those disclosed in U.S. Patent Application Serial No. 15 / 329,683 or International Patent Application Publication No. WO 2016 / 018960, and the entire contents of each application are incorporated herein by reference.

[0065] In one set of embodiments, determination of the genotype of a cell can be facilitated by determining an identification portion of a nucleic acid within the cell. For example, a nucleic acid comprising an identification portion and a guide portion can be introduced into a cell; the guide portion can result in the formation of different phenotypes as discussed above, for example, by allowing editing of a target sequence to occur, for example, editing occurs on the genome. However, it is also important to know which cells the nucleic acid is introduced into, so that the relationship between the observed phenotype (e.g., an altered phenotype) and the genotype that causes those phenotypes can be understood. As discussed herein, by determining the identification portion within the cell, the identity of the nucleic acid contained within each cell can be determined, thereby also determining a specific guide portion, for example, if the nucleic acid comprises an identification portion and a guide portion on the same single nucleic acid.

[0066] As a non-limiting example, in one set of embodiments, the cell can be sequentially exposed to nucleic acid probes capable of binding to different portions of the identification portion or to molecules (such as RNA) expressed by the cell from the identification portion, for example, the nucleic acid probe is a nucleic acid probe comprising a target sequence (e.g., particularly in some cases, which is capable of binding to at least a portion of the identification portion) and a reading sequence (e.g., which can be "read" in some manner to determine binding); and the binding of the nucleic acid probe within the cell can be determined. For example, the cell can be exposed to a secondary nucleic acid probe, which can include a recognition sequence capable of binding or hybridizing to a reading sequence, and can include a signaling entity. By determining the signaling entity within an image (and in some cases, inactivating the signaling entity between images and exposing to a different nucleic acid probe), the identification portion of the cell can be determined.

[0067] As discussed herein, various nucleic acid probes can be used to determine one or more nucleic acids in a cell. The probe can include nucleic acids (or entities that can be hybridized with nucleic acids, for example, specific hybridization), such as DNA, RNA, LNA (locked nucleic acid), PNA (peptide nucleic acid), or a combination thereof. In some cases, other components may also be present in the nucleic acid probe, for example, as discussed below. In some embodiments, the nucleic acid probe may be formed by other components, for example, proteins or other small molecules, or may represent a combination of these components and nucleic acids (such as DNA, RNA, LNA, PNA, etc.).

[0068] Nucleic acid probes can be introduced into cells using any suitable method. In some cases, the cells can be sufficiently permeabilized so that the nucleic acid probe can be introduced into the cells by flowing a fluid containing the nucleic acid probe around the cells. In some cases, the cells can be sufficiently permeabilized as part of a fixation process; in other embodiments, cells can be sufficiently permeabilized by exposure to certain chemicals such as ethanol, methanol, Triton, etc. In addition, in some embodiments, techniques such as electroporation or microinjection can be used to introduce nucleic acid probes into cells.

[0069] Determination of intracellular nucleic acid can be qualitative and / or quantitative. In addition, the determination can also be spatial, for example, the position of intracellular nucleic acid can be determined in two dimensions or three dimensions. In some embodiments, the position, number and / or concentration of intracellular nucleic acid can be determined.

[0070] As mentioned, in certain embodiments, the reporter gene position and the association or co-location between the detection read sequence when reading the codeword can greatly improve the decoding accuracy, for example, due to the reduction of the error identification of the background signal introduced by the non-specific label. In some cases, for example, the codeword readout portion can only include a sequence for reading, so that the readout signal can be more difficult to identify, for example, relative to the background. For example, in some cases, the reporter gene portion can be determined as discussed herein, for example, locally or spatially, and the portion of the identification sequence can be determined as discussed herein. In some cases, the apparent portion of the identification sequence that is not co-located with the reporter gene portion can be eliminated from further consideration. For example, the apparent identification sequence can be an incorrect signal, background noise, etc. In addition, in some cases, the reporter gene portion can be determined between different determinations of the identification sequence. Such a mode can improve accuracy, for example, reducing the error caused by the movement of the sample, the platform offset, etc. Therefore, the association or co-location between the reporter gene position and the detection read sequence can be used to determine whether the assumed signal of the read sequence is a read sequence or background noise, etc. (therefore not worthy of further consideration).

[0071] It will be appreciated that while the number of guide moieties and / or identifier moieties can have a relatively large number of possibilities (e.g., millions), this is readily accomplished by one of ordinary skill in the art using techniques such as computers and automated nucleic acid synthesis instruments (many of which are commercially available) and techniques such as solid phase synthesis and / or isothermal assembly, and / or error-prone PCR and / or ligation or otherwise assembly by, for example, performing overlapping PCR on multiple variable regions in combination. Similarly, a correspondingly relatively large number of unique identifier moieties can be associated with such a large number of possibilities for guide moieties, for example, by using a relatively small number of appropriate variable regions and a unique "position" that can be generated for each variable region. Thus, nucleic acid libraries (e.g., each containing an identifier moiety and a guide moiety) can be prepared, for example, containing at least 10, at least 10 2 At least 10 3 At least 10 4 At least 10 5 At least 10 6 At least 10 7 At least 10 8 The only member.

[0072] In some aspects, nucleic acid from a nucleic acid library can be introduced into a cell. Any suitable technique can be used to introduce the nucleic acid. For example, in one group of embodiments, nucleic acid can be delivered to a cell using a virus, such as a slow virus, a retrovirus, an adenovirus, or an adeno-associated virus. In some cases, the virus can be capable of transfecting or delivering the nucleic acid to the genome of the cell, and in some cases, stably in the genome.

[0073] For example, in one set of embodiments, a lentiviral delivery system can be used to introduce nucleic acids into cells. The lentiviral system can allow a certain number of nucleic acids to be introduced into cells to be controlled. For example, by controlling the titer of the lentivirus used for transduction, the number of library members delivered to a single cell can be controlled to be 1, or more than 1. In some embodiments, the guide portion and the identification portion can be placed in close proximity to each other in the 3'LTR region of the lentivirus, that is, after the polypurine band (PPT) sequence of the lentivirus, so that the distance between the guide portion and the identification portion is minimized, for example, for the constant region of the sgRNA of Cas9, it is 100 bases or less. In certain embodiments, the distance can also be less than 500 bases, less than 300 bases, less than 200 bases, less than 100 bases, less than 50 bases, less than 30 bases, or less than 10 bases. Such a lentiviral construct can reduce the genomic distance between the guide portion and the identification portion. This can lead to a reduction in the recombination effect, which can allow the guide portion to be more accurately identified by measuring the identification portion. Those of ordinary skill in the art are familiar with lentiviral and other viral-based delivery systems for introducing nucleic acids into cells. Many kits capable of such delivery of nucleic acids into cells using viruses are readily commercially available.

[0074] In addition, in some embodiments, other techniques can be used to introduce nucleic acids into cells. For example, the nucleic acid can be incorporated into a plasmid, which can be taken up by the cell. Other methods for introducing nucleic acids into cells include, but are not limited to, calcium phosphate (e.g., tricalcium phosphate), electroporation, cell extrusion, mixing cationic lipids with materials that produce liposomes (liposomes fuse with cell membranes), etc. Other non-limiting examples of suitable methods include dendrimers, cationic polymers, lipofection, FuGENE, sonoporation, optical transfection, protoplast fusion, impalefection, gene guns, magnetic transfection, particle bombardment, viral infection, etc.

[0075] In certain embodiments, the nucleic acid can be introduced or transfected into the cell so that at least 50% of the cells only introduce 0 or 1 nucleic acid therein. In some cases, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% of the cells can only introduce 0 and / or 1 nucleic acid therein. This can be, for example, using slow viruses such as those described above, suitable dilution techniques, cell sorting techniques, or by using other techniques such as microfluidic droplets to achieve. In other cases, the percentage of transfected cells can be smaller, such as less than 50%, less than 20%, less than 10%, less than 1%. In some embodiments, cells in which these nucleic acids are not introduced can be removed. Non-limiting examples of cell removal include treatment with chemicals (such as antibiotics), for example, killing untransfected cells or preventing untransfected cells from dividing. In another example, some or all of the cells not containing the introduced nucleic acid can be removed from the sample, for example, using fluorescence activated cell sorting and / or other suitable cell sorting or microfluidic techniques.

[0076] In certain embodiments, the identification portion and the guide portion can be combined in a single source, e.g., a nucleic acid contained in a single virus. In other embodiments, these portions can be provided to the cell in separate sources, e.g., two different viral delivery vehicles. Other examples of introducing nucleic acids into cells are disclosed herein, and the method of introduction can be the same or different.

[0077] The merging of the identification portion and the guide portion can be determined, whether on the same or different carriers (e.g., viruses), for example, randomly or deterministically. For example, a specified CRISPR edit can be assigned to a specified barcode and expressed in a cell. In some embodiments, the specific association between the identification portion and the guide portion can be measured using any of a variety of techniques. For example, PCR can be used to amplify a nucleic acid portion containing both the identification portion and the guide portion, and then sequencing methods, including next-generation sequencing, can be used to identify which identification region and which guide portion appear by direct sequencing of this PCR product. A person of ordinary skill in the art is aware of other techniques that can be used to sequence nucleic acids (e.g., containing the identification portion and the guide portion). Any technology can be used for sequencing, for example, Sanger sequencing, high-throughput sequencing, next-generation sequencing, nanopore sequencing, ligation sequencing, synthesis sequencing, etc. A person of ordinary skill in the art is aware of different techniques for sequencing nucleic acids.

[0078] Cells can be analyzed in some aspects to determine their phenotypes. In some cases, in some embodiments, the phenotype can be changed, for example, by using CRISPR or other techniques, for example, the genome of the cell can be interacted with, as discussed herein. Any suitable technique can be used to determine the phenotype, for example, using optical techniques, by analyzing cell behavior, etc. Specific examples include, but are not limited to, microscopes or other optical techniques such as optical microscopes, fluorescence microscopes, confocal microscopes, near-field microscopes, two-photon microscopes, or phase contrast microscopes, or other techniques described herein. In some cases, super-resolution techniques can be used, including any of those described herein. In some embodiments, phenotypes can be detected by other techniques, such as atomic force microscopy or patch clamps. In addition, in some embodiments, proteins can be used to determine phenotypes. For example, proteins can be determined using fluorescence, immunofluorescence, etc. Specific non-limiting examples include fluorescent labeling methods such as fluorescent proteins or organic dyes. In some cases, microscopes and another technology can be used in combination to determine phenotypes.

[0079] Examples of phenotypes that can be determined include, but are not limited to, the morphology of the cell (e.g., shape, size, visual appearance, organelles, subcompartments, states (e.g., during the cell cycle), etc.), certain characteristics of cell movement (e.g., speed, persistence, chemotactic behavior, etc.), certain characteristics of cell-cell interactions (e.g., cell-cell adhesion, cell-cell avoidance, cell-cell interactions, etc.), or certain subcellular characteristics (e.g., location of proteins or nucleic acids, diffusion of proteins or nucleic acids, binding of two or more proteins and / or nucleic acids, etc.). The morphology can include whole cell morphology or subcompartment morphology. In one embodiment, smFISH is used to determine the phenotype of the cell.

[0080] In some cases, the phenotype can be determined dynamically, e.g., over time in a cell.

[0081] In certain embodiments, the cells are present on a substrate, e.g., a substrate suitable for culturing and / or imaging cells. For example, the substrate can be glass, silicon, plastic (e.g., polystyrene, polypropylene, polycarbonate, etc.), or the like. In some cases, at least a portion of the substrate can be at least partially light-transmissive. The substrate can also be untreated or treated in some manner to promote cell attachment.

[0082] In some embodiments, the phenotype that can be determined includes all or at least a portion of the transcriptome of the cell. A variety of techniques can be used to determine the transcriptome, including, but not limited to, smFISH, MERFISH or other techniques such as those described herein. Also referring to U.S. Patent Application Serial No. 15 / 329,683 or International Patent Application Publication No. WO 2016 / 018960, the entire contents of each are incorporated herein by reference. In some cases, the transcriptome can be determined spatially in one or more cells.

[0083] In addition, in some cases, the phenotype that can be determined includes all or at least a portion of the chromosome of the cell, and / or an agent such as protein or RNA that can be combined with the chromosome of the cell or associate in another way. For example, the concentration, spatial position, activity, association of chromosome and / or other associated agents, etc. can be determined according to some embodiments of the present invention. In some cases, chromosome can be determined spatially in one or more cells. The non-limiting examples of the technology that can be used to determine chromosome include multiple DNA FISH or CASFISH. As another example, the epigenetic modification of the cell can be determined.

[0084] Additionally, in some cases, the phenotype that can be determined includes all or at least a portion of the proteome of the cell. A variety of techniques can be used to determine the proteome, including antibody labeling, sequential antibody labeling, multiplex antibody imaging, or other multiplex protein imaging techniques. For example, the concentration, spatial location, activity, association, etc. of proteins and / or other associated agents can be determined.

[0085] In certain embodiments, one or more markers in a cell can be determined to determine the phenotype. For example, the marker can indicate a certain cell protein, nucleic acid, morphological characteristics, etc., or the marker can indicate cell behavior. In addition, the marker can be a marker that can be visually determined in some cases. For example, the marker can be fluorescent, or can change the fluorescence of another fluorescent entity in the cell (for example, by enhancing or quenching). In some embodiments, the marker can also be a dye or can change color. Therefore, the difference in intensity, wavelength, frequency, position, distribution, etc. between cells in the image can be determined to determine the phenotype of the cell. In some cases, other methods for determining markers can also be used; for example, the marker can be radioactive. Many such markers are commercially available.

[0086] In addition, it should be understood that these measurements are not mutually exclusive. Any combination of these measurements can be carried out in a single sample. And in some embodiments such measurement can be repeated, for example, for the same sample. For example, the measurement can be repeated to ensure correctness or reduce potential errors (for example, measurement errors), or after being exposed to various stimuli or conditions, the measurement is repeated, such as with different nutritional sources, small molecules or other suitable agents that can interact with cells.

[0087] In some cases, the phenotype of a cell can be changed by applying a guide portion, for example, as discussed above, the guide portion can be expressed by the cell in some form to change its phenotype. For example, a guide portion can be used to induce changes in the genome of a cell, for example, by CRISPR or other suitable techniques, including those described herein. As another example, a guide portion encoding a protein can be added to a cell, and the cell can express the protein. If different proteins are encoded in different cells, the cell can exhibit different phenotypes, which can be determined as described above. Therefore, for example, a plurality of cells can be transfected or otherwise introduced with a plurality of different guide portions, and the cells are then studied to determine the effects of the different guide portions on their phenotypes.

[0088] Therefore, some aspects generally relate to nucleic acid probes introduced into cells (or other samples). The probe may include any of a variety of entities that can hybridize with nucleic acids (e.g., target sites), typically by Watson-Crick base pairing, such as DNA, RNA, LNA, PNA, etc., depending on the application. Nucleic acid probes typically include a target sequence that can be combined with at least a portion of a target (e.g., target site). In some cases, the combination can be specific binding (e.g., by complementary binding). When introduced into cells or other systems, the target sequence can be combined with a specific target (e.g., mRNA, or other nucleic acids, as discussed herein). The nucleic acid probe may also include one or more read sequences, as discussed below.

[0089] In some cases, more than one type of nucleic acid probe can be applied to the sample, for example, successively or simultaneously. For example, at least 2, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 300, at least 1,000, at least 3,000, at least 10,000 or at least 30,000 distinguishable nucleic acid probes can be applied to the sample. In some cases, the nucleic acid probes can be added successively. However, in some cases, more than one nucleic acid probe can be added simultaneously.

[0090] The nucleic acid probe may include one or more target sequences, which may be located at any position within the nucleic acid probe. The target sequence may include a region that is substantially complementary to a portion of a target, such as a target nucleic acid. For example, in some cases, the portion may be at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary, for example, to produce specific binding. Typically, complementarity is determined based on Watson-Crick nucleotide base pairing.

[0091] In some cases, the target sequence can be at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 65, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 nucleotides in length. In some cases, the target sequence may be no more than 500, no more than 450, no more than 400, no more than 350, no more than 300, no more than 250, no more than 200, no more than 175, no more than 150, no more than 125, no more than 100, no more than 75, no more than 60, no more than 65, no more than 60, no more than 55, no more than 50, no more than 45, no more than 40, no more than 35, no more than 30, no more than 20, or no more than 10 nucleotides in length. Any combination of these is also possible, for example, the target sequence may have a length of 10-30 nucleotides, 20-40 nucleotides, 5-50 nucleotides, 10-200 nucleotides, or 25-35 nucleotides, 10-300 nucleotides, etc.

[0092] The target sequence of nucleic acid probe can be determined with reference to the target suspected to be present in the cell or other sample.For example, the target nucleic acid of protein can be determined using the sequence of protein, for example, by determining the nucleic acid that forms the protein by expression.In some cases, only a part of the nucleic acid encoding the protein is used, for example, with a length as discussed above.In addition, in some cases, more than one target sequence that can be used to identify a specific target can be used.For example, multiple probes can be used, and they can be combined or hybridized with the same or different regions of the same target one after another and / or simultaneously.Hybridization typically refers to an annealing process, by which the complementary single-stranded nucleic acids are associated to form double-stranded nucleic acids by Watson-Crick nucleotide base pairing (for example, hydrogen bonding, guanine-cytosine and adenine-thymine).

[0093] In some embodiments, nucleic acid probes can also include one or more "reading" sequences, as described above. The reading sequence can be used to identify the nucleic acid probe, for example, by associating with a signaling entity, as described below. In some embodiments, the nucleic acid probe can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more, 20 or more, 24 or more, 32 or more, 40 or more, 48 or more, 50 or more, 64 or more, 75 or more, 100 or more, 128 or more reading sequences. The reading sequence can be located at any position in the nucleic acid probe. If there is more than one reading sequence, the reading sequence can be located next to each other, and / or spaced with other sequences.

[0094] The read sequence can be of any length. If more than one read sequence is used, the read sequences can independently have the same or different lengths. For example, the read sequence can be at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 65, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 nucleotides in length. In some cases, the length of the read sequence can be no more than 500, no more than 450, no more than 400, no more than 350, no more than 300, no more than 250, no more than 200, no more than 175, no more than 150, no more than 125, no more than 100, no more than 75, no more than 60, no more than 65, no more than 60, no more than 55, no more than 50, no more than 45, no more than 40, no more than 35, no more than 30, no more than 20, or no more than 10 nucleotides. Any combination of these is also possible, for example, the read sequence can have a length of 10-30 nucleotides, 20-40 nucleotides, 5-50 nucleotides, 10-200 nucleotides, or 25-35 nucleotides, 10-300 nucleotides, etc.

[0095] In some embodiments, the read sequence can be arbitrary or random. In some cases, the read sequence can be selected to reduce or minimize the homology with other components of the cell or other samples, for example, so that the read sequence itself is not combined or hybridized with other nucleic acids suspected in the cell or other samples. In some cases, homology can be less than 10%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In some cases, there can be less than 20 base pairs, less than 18 base pairs, less than 15 base pairs, less than 14 base pairs, less than 13 base pairs, less than 12 base pairs, less than 11 base pairs, or less than 10 base pairs of homology. In some cases, such base pairs are continuous.

[0096] In one set of embodiments, a set of nucleic acid probes may include a specific number of read sequences, which in some cases may be less than the number of targets of the nucleic acid probes. One of ordinary skill in the art will appreciate that if there is one signaling entity and n read sequences, then generally 2 can be uniquely identified. n-1 different nucleic acid targets. However, it is not necessary to use all possible combinations. For example, a group of nucleic acid probes can target 12 different nucleic acid sequences, but contain no more than 8 read sequences. As another example, a group of nucleic acids can target 140 different nucleic acid species, but contain no more than 16 read sequences. Different nucleic acid sequence targets can be identified individually by using different combinations of read sequences in each probe. For example, each probe can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, etc. or more read sequences. In some cases, a group of nucleic acid probes can each contain the same number of read sequences, although in other cases, different numbers of read sequences can be present on various probes.

[0097] As a non-limiting example, a first nucleic acid probe may comprise a first target sequence, a first read sequence, and a second read sequence, while a second, different nucleic acid probe may comprise a second target sequence, the same first read sequence, but a third read sequence replaces the second read sequence. Such probes can thus be distinguished by determining the various read sequences present or associated with a given probe or position, as discussed herein. For example, the probes can be continuously identified and encoded using "code words," as discussed below. Optionally, the code words can also be subject to error detection and / or correction.

[0098] In addition, in certain embodiments, a set of nucleic acid probes (and their corresponding complementary sites on the encoded probes) can be prepared using only 2 or only 3 of the 4 natural nucleotide bases, such as excluding all "G" or excluding all "C" within the set of probes. In certain embodiments, sequences lacking "G" or "C" can form very little secondary structure and can promote more uniform and faster hybridization. Thus, in some cases, the nucleic acid probes can contain only A, T, and G; only A, T, and C; only A, C, and G; or only T, C, and G.

[0099] In one aspect, the read sequence on the nucleic acid probe can be able to bind to the corresponding recognition sequence on the primary amplification nucleic acid (e.g., specifically bind). Therefore, when the nucleic acid probe identifies a target (e.g., a DNA or RNA target) in a biological sample, the primary amplification nucleic acid can also associate with the target via the nucleic acid probe using the interaction (e.g., complementary binding) between the read sequence of the nucleic acid probe and the corresponding recognition sequence on the primary amplification nucleic acid. For example, the recognition sequence can recognize the target read sequence, but does not substantially recognize or bind to other non-target read sequences. The primary amplification nucleic acid can also include any one of a variety of entities that can hybridize with nucleic acids, for example, DNA, RNA, LNA, and / or PNA, etc., depending on the application. For example, these entities can form some or all of the recognition sequence. Therefore, the recognition sequence can recognize a nucleic acid sequence, such as DNA or RNA.

[0100] In some cases, the recognition sequence can be substantially complementary to the target read sequence. In some cases, the sequence can be at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary. Typically, complementarity is determined based on Watson-Crick nucleotide base pairing. The structure of the target read sequence can include those described above.

[0101] In some cases, the recognition sequence can be at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 65, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 nucleotides in length. In some cases, the length of the recognition sequence can be no more than 500, no more than 450, no more than 400, no more than 350, no more than 300, no more than 250, no more than 200, no more than 175, more than 150, no more than 125, no more than 100, no more than 75, no more than 60, no more than 65, no more than 60, no more than 55, no more than 50, no more than 45, no more than 40, no more than 35, no more than 30, no more than 20, or no more than 10 nucleotides. Any combination of these is also possible, for example, the recognition sequence can have a length of 10-30 nucleotides, 20-40 nucleotides, 5-50 nucleotides, 10-200 nucleotides, or 25-35 nucleotides, 10-300 nucleotides, etc.

[0102] In some embodiments, primary amplification nucleic acid can also include one or more read sequences that can be combined with secondary amplification nucleic acid, as described below.For example, primary amplification nucleic acid can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more, 20 or more, 32 or more, 40 or more, 50 or more, 64 or more, 75 or more, 100 or more, 128 or more read sequences.The read sequence can be positioned at any position in primary amplification nucleic acid.If there is more than one read sequence, the read sequence can be located next to each other, and / or spaced with other sequences.In one embodiment, the primary amplification nucleic acid includes a recognition sequence at the first end and a plurality of read sequences at the second end.

[0103] In some cases, the length of the read sequence within the primary amplified nucleic acid can be at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 65, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 nucleotides. In some cases, the length of the read sequence can be no more than 500, no more than 450, no more than 400, no more than 350, no more than 300, no more than 250, no more than 200, no more than 175, no more than 150, no more than 125, no more than 100, no more than 75, no more than 60, no more than 65, no more than 60, no more than 55, no more than 50, no more than 45, no more than 40, no more than 35, no more than 30, no more than 20, or no more than 10 nucleotides. Combinations of any of these are also possible, for example, the read sequence can have a length of 10-20 nucleotides, 10-30 nucleotides, 20-40 nucleotides, 5-50 nucleotides, 10-200 nucleotides, or 25-35 nucleotides, 10-300 nucleotides, etc.

[0104] Any number of read sequences may be present in the primary amplified nucleic acid. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more read sequences may be present in the primary amplified nucleic acid. If more than one read sequence is present in the primary amplified nucleic acid, the read sequences may be the same or different. In some cases, for example, the read sequences may all be the same.

[0105] In some embodiments, a set of primary amplified nucleic acids can be prepared using only 2 or only 3 of the 4 natural nucleotide bases, such as excluding all "G" or excluding all "C" within the set of nucleic acids. In certain embodiments, sequences lacking "G" or "C" can form very few secondary structures and can promote more uniform and faster hybridization. Thus, in some cases, the primary amplified nucleic acids can contain only A, T, and G; only A, T, and C; only A, C, and G; or only T, C, and G.

[0106] In some cases, more than one type of primary amplification nucleic acid can be applied to a sample, e.g., sequentially or simultaneously. For example, at least 2, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 300, at least 1,000, at least 3,000, at least 10,000, or at least 30,000 distinguishable primary amplification nucleic acids can be applied to a sample. In some cases, the primary amplification nucleic acids can be added sequentially. However, in some cases, more than one primary amplification nucleic acid can be added simultaneously.

[0107] In one group of embodiments, the read sequence on the primary amplified nucleic acid can be capable of binding to (e.g., specifically binding to) a corresponding recognition sequence on a secondary amplified nucleic acid. Therefore, when a nucleic acid probe recognizes a target (e.g., a DNA or RNA target) in a biological sample, the secondary amplified nucleic acid can also associate with the target via the primary amplified nucleic acid using the read sequence of the primary amplified nucleic acid and the interaction (e.g., complementary binding) between the corresponding recognition sequence on the secondary amplified nucleic acid. For example, the recognition sequence on the secondary amplified nucleic acid can recognize the read sequence on the primary amplified nucleic acid, but does not substantially recognize or bind to other non-target read sequences. The secondary amplified nucleic acid can also include any of a variety of entities capable of hybridizing with nucleic acids, such as DNA, RNA, LNA, and / or PNA, etc., depending on the application. For example, these entities can form some or all of the recognition sequences.

[0108] In some cases, the recognition sequence on the secondary amplified nucleic acid can be substantially complementary to the read sequence on the primary amplified nucleic acid. In some cases, the sequence can be at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary.

[0109] In some cases, the length of the recognition sequence on the secondary amplified nucleic acid can be at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 65, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 nucleotides. In some cases, the length of the recognition sequence can be no more than 500, no more than 450, no more than 400, no more than 350, no more than 300, no more than 250, no more than 200, no more than 175, no more than 150, no more than 125, no more than 100, no more than 75, no more than 60, no more than 65, no more than 60, no more than 55, no more than 50, no more than 45, no more than 40, no more than 35, no more than 30, no more than 20, or no more than 10 nucleotides. Any combination of these is also possible, for example, the recognition sequence can have a length of 10-30 nucleotides, 20-40 nucleotides, 5-50 nucleotides, 10-200 nucleotides, or 25-35 nucleotides, 10-300 nucleotides, etc.

[0110] In some embodiments, the secondary amplified nucleic acid can also include one or more read sequences that can bind to a signaling entity, as discussed herein. For example, the secondary amplified nucleic acid can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more, 20 or more, 32 or more, 40 or more, 50 or more, 64 or more, 75 or more, 100 or more, 128 or more read sequences that can bind to a signaling entity. The read sequence can be located at any position in the secondary amplified nucleic acid. If there is more than one read sequence, the read sequence can be located next to each other, and / or spaced with other sequences. In one embodiment, the secondary amplified nucleic acid includes a recognition sequence at the first end and a plurality of read sequences at the second end. This structure can also be the same or different from the structure of the primary amplified nucleic acid.

[0111] In some cases, the length of the read sequence within the secondary amplified nucleic acid can be at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 65, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 nucleotides. In some cases, the length of the read sequence can be no more than 500, no more than 450, no more than 400, no more than 350, no more than 300, no more than 250, no more than 200, no more than 175, no more than 150, no more than 125, no more than 100, no more than 75, no more than 60, no more than 65, no more than 60, no more than 55, no more than 50, no more than 45, no more than 40, no more than 35, no more than 30, no more than 20, or no more than 10 nucleotides. Combinations of any of these are also possible, for example, the read sequence within the secondary amplified nucleic acid can have a length of 10-20 nucleotides, 10-30 nucleotides, 20-40 nucleotides, 5-50 nucleotides, 10-200 nucleotides, or 25-35 nucleotides, 10-300 nucleotides, etc.

[0112] Any number of read sequences can be present in the secondary amplified nucleic acid. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more read sequences can be present in the secondary amplified nucleic acid. If there are more than one read sequence in the secondary amplified nucleic acid, the read sequences can be the same or different. In some cases, for example, the read sequences can all be the same. In addition, the same or different number of read sequences can be independently present in the primary amplified nucleic acid and the secondary amplified nucleic acid.

[0113] The set of secondary amplified nucleic acids can be prepared using only 2 or only 3 of the 4 natural nucleotide bases, in certain embodiments, such as excluding all "G" or excluding all "C" within the set of nucleic acids. In certain embodiments, sequences lacking "G" or "C" can form very little secondary structure and can promote more uniform and faster hybridization. Thus, in some cases, the secondary amplified nucleic acids can contain only A, T, and G; only A, T, and C; only A, C, and G; or only T, C, and G.

[0114] In some cases, more than one type of secondary amplified nucleic acid can be applied to a sample, e.g., sequentially or simultaneously. For example, at least 2, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 300, at least 1,000, at least 3,000, at least 10,000, or at least 30,000 distinguishable secondary amplified nucleic acids can be applied to a sample. In some cases, the secondary amplified nucleic acids can be added sequentially. However, in some cases, more than one secondary amplified nucleic acid can be added simultaneously.

[0115] In addition, in certain embodiments, this pattern can be repeated before the signaling entity, for example, using tertiary amplified nucleic acids, quaternary nucleic acids, etc., similar to the discussion above. Thus, the signaling entity can be combined with the last amplified nucleic acid. Thus, as a non-limiting example, the encoding nucleic acid probe can be combined with the target, the primary amplified nucleic acid can be combined with the encoding nucleic acid probe, the secondary amplified nucleic acid can be combined with the primary amplified nucleic acid, the tertiary amplified nucleic acid can be combined with the secondary amplified nucleic acid, and the signaling entity can be combined with the tertiary amplified nucleic acid, or the encoding nucleic acid probe can be combined with the target, the primary amplified nucleic acid can be combined with the encoding nucleic acid probe, the secondary amplified nucleic acid can be combined with the primary amplified nucleic acid, the tertiary amplified nucleic acid can be combined with the secondary amplified nucleic acid, the quaternary amplified nucleic acid can be combined with the tertiary amplified nucleic acid, and the signaling entity can be combined with the quaternary amplified nucleic acid, etc. Therefore, in all embodiments, the last amplified nucleic acid need not necessarily be the secondary amplified nucleic acid.

[0116] In some aspects, cells can be immobilized or fixed to a substrate, e.g., prior to determining the genotype, as discussed below. In some cases, the immobilization or fixation of the cells can be performed after the phenotype is determined. This can be useful according to certain embodiments, e.g., for correlating the phenotype of a cell within an image with the genotype of a subsequent cell (e.g., determined as discussed below). In some embodiments, the cells can also be fixed prior to measuring the phenotype, rather than after measuring the phenotype and before measuring the genotype.

[0117] Those of ordinary skill in the art know systems and methods for fixing cells on substrates or otherwise immobilizing cells. As non-limiting examples, cells can be fixed using chemicals such as formaldehyde, paraformaldehyde, glutaraldehyde, ethanol, methanol, acetone, acetic acid, etc. In one embodiment, cells can be fixed using an organic solvent (HOPE) mediated by Hepes-glutamate buffer. Also referring to U.S. Patent Application Serial No. 62 / 419,033, the entire contents of which are incorporated herein by reference.

[0118] Certain aspects of the invention relate to assay samples, which may include cell cultures, cell suspensions, biological tissues, biopsies, organisms, and the like. In some cases, the sample may also be cell-free but contain nucleic acids. If the sample contains cells, the cells may be human cells, or any other suitable cells, e.g., mammalian cells, fish cells, insect cells, plant cells, and the like. In some cases, more than one type of cell may be present.

[0119] In a sample, the target to be determined may include nucleic acids, proteins, and the like. The nucleic acids to be determined may include, for example, DNA (e.g., genomic DNA), RNA, or other nucleic acids present in a cell (or other sample). The nucleic acid may be endogenous to the cell, or may be added to the cell. For example, the nucleic acid may be a viral nucleic acid, or may be artificially established. In some cases, the nucleic acid to be determined may be expressed by the cell. In some embodiments, the nucleic acid is RNA. The RNA may be coded and / or non-coded RNA. For example, the RNA may encode a protein. Non-limiting examples of RNA that can be studied in a cell include mRNA, siRNA, rRNA, miRNA, tRNA, lncRNA, snoRNA, snRNA, exRNA, piRNA, and the like.

[0120] In some cases, a majority of nucleic acids in a cell can be studied. For example, in some cases, sufficient RNA present in a cell can be determined to generate a partial or complete transcriptome of the cell. In some cases, at least 4 types of mRNA are determined in a cell, and in some cases, at least 3, at least 4, at least 7, at least 8, at least 12, at least 14, at least 15, at least 16, at least 22, at least 30, at least 31, at least 32, at least 50, at least 63, at least 64, at least 72, at least 75, at least 100, at least 127, at least 128, at least 140, at least 255, at least 256, at least 50, at least 63, at least 64, at least 72, at least 75, at least 100, at least 127, at least 128, at least 140, at least 255, at least 256, at least 50 At least 10, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 4,000, at least 5,000, at least 7,500, at least 10,000, at least 12,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 40,000, at least 50,000, at least 75,000, or at least 100,000 types of mRNA.

[0121] In some cases, the transcriptome of the cell can be determined. It should be understood that the transcriptome generally encompasses all RNA molecules produced in the cell, not just mRNA. Therefore, for example, in some cases, the transcriptome can also include rRNA, tRNA, siRNA, etc. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the cell transcriptome can be determined.

[0122] In some embodiments, other targets to be determined can include targets connected to nucleic acids, proteins, etc. For example, in one group of embodiments, a binding entity capable of recognizing a target can be conjugated to a nucleic acid probe. The binding entity can be any entity that can recognize a target (e.g., specific or non-specific recognition). Non-limiting examples include enzymes, antibodies, receptors, complementary nucleic acid chains, aptamers, etc. For example, an antibody connected to an oligonucleotide can be used to determine a target. The target can be bound to an antibody connected to an oligonucleotide, and the oligonucleotide is determined as discussed herein.

[0123] The determination of targets such as nucleic acids in cells or other samples can be qualitative and / or quantitative. In addition, the determination can also be spatial, for example, the position of nucleic acids or other targets in cells or other samples can be determined in two dimensions or three dimensions. In some embodiments, the position, number and / or concentration of nucleic acids or other targets in cells or other samples can be determined.

[0124] In some cases, a majority of the genome of a cell can be assayed. The genomic segments assayed can be genomically continuous or discontinuous. For example, in some cases, at least 4 genomic segments within a cell are assayed, and in some cases, at least 3, at least 4, at least 7, at least 8, at least 12, at least 14, at least 15, at least 16, at least 22, at least 30, at least 31, at least 32, at least 50, at least 63, at least 64, at least 72, at least 75, at least 100, at least 127, at least 128, at least 140, at least 255, at least 256, at least 257, at least 261, at least 262, at least 263, at least 264, at least 265, at least 266, at least 267, at least 268, at least 269, at least 270, at least 271, at least 272, at least 273, at least 274, at least 275, at least 276, at least 277, at least 278, at least 279, at least 280, at least 281, At least 10,000, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 4,000, at least 5,000, at least 7,500, at least 10,000, at least 12,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 40,000, at least 50,000, at least 75,000, or at least 100,000 genomic segments.

[0125] In some cases, the entire genome of the cell can be determined, and it should be understood that the genome generally covers all DNA molecules produced in the cell, not just chromosomal DNA. Thus, for example, in some cases, for example, in addition to (or instead of) chromosomal DNA, the genome can also include mitochondrial DNA, chloroplast DNA, plasmid DNA, etc. In some embodiments, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or 100% of the genome of the cell can be determined.

[0126] As discussed herein, according to some aspects, a variety of nucleic acid probes can be used to measure one or more targets in cells or other samples. The probe can include nucleic acid (or an entity that can be hybridized with nucleic acids, for example, an entity of specific hybridization), such as DNA, RNA, LNA (locked nucleic acid), PNA (peptide nucleic acid), and / or a combination thereof. In some cases, other components may also be present in the nucleic acid probe, for example, as discussed herein. In addition, any suitable method can be used to introduce the nucleic acid probe into the cell.

[0127] Other components may also be present in the nucleic acid probe or may also be present in the amplified nucleic acid. For example, in one group of embodiments, one or more primer sequences may be present, for example, to promote enzyme amplification. One of ordinary skill in the art knows primer sequences suitable for applications such as amplification (for example, using PCR or other suitable techniques). Many such primer sequences are commercially available. Other examples of sequences that may be present in the primary nucleic acid probe include, but are not limited to, promoter sequences, operators, identifier sequences, nonsense sequences, etc.

[0128] Typically, primer is a single-stranded or partially double-stranded nucleic acid (e.g., DNA), and primer serves as the starting point for nucleic acid synthesis, allowing polymerase such as nucleic acid polymerase to extend the primer and replicate the complementary strand. Primer (e.g., designed to) is complementary to the target nucleic acid and hybridizes. In some embodiments, primer is a synthetic primer. In some embodiments, primer is a non-natural primer. Primer typically has a length of 10-50 nucleotides. For example, primer can have a length of 10-40, 10-30, 10-20, 25-50, 15-40, 15-30, 20-50, 20-40 or 20-30 nucleotides. In some embodiments, the length of primer is 18-24 nucleotides.

[0129] In some embodiments, one or more signaling entities can be combined with a recognition entity on a secondary amplified nucleic acid (or other final amplified nucleic acid). Non-limiting examples of signaling entities include fluorescent entities (fluorophores) or phosphorescent entities, for example, as discussed below. The signaling entity can then be determined, for example, to determine the nucleic acid probe or the target. In some cases, the determination can be spatial, for example, two-dimensionally or three-dimensionally. In addition, in some cases, the determination can be quantitative, for example, the amount or concentration of the signaling entity and / or the target can be determined.

[0130] In one set of embodiments, the signaling entity can be attached to the secondary amplified nucleic acid (or other last amplified nucleic acid). The signaling entity can be attached to the secondary amplified nucleic acid (or other last amplified nucleic acid) before or after the secondary amplified nucleic acid associates with the target in the sample. For example, the signaling entity can be attached to the secondary amplified nucleic acid initially or after the secondary amplified nucleic acid has been applied to the sample. In some cases, the signaling entity is added and then the signaling entity reacts to attach it to the amplified nucleic acid.

[0131] In one group of embodiments, the signaling entity can be attached to the nucleotide sequence by a key, and the key can be cleaved to release the signaling entity. For example, after determining the distribution of nucleic acid probes in the sample, the signaling entity can be released or inactivated, followed by another round of nucleic acid probes and / or amplified nucleic acids. Therefore, in some embodiments, the key can be a cleavable key, such as a disulfide bond or a photocleavable key. Examples of photocleavable keys are discussed in detail herein. In some cases, such a key can be cleaved, for example, after being exposed to a reducing agent or light (e.g., ultraviolet light). Other details are described below. Other examples of systems and methods for inactivating and / or removing signaling entities are discussed in more detail herein.

[0132] In certain embodiments, the use of primary and secondary amplified nucleic acids suggests that there is a maximum number of signaling entities that can bind to a specified nucleic acid probe. For example, there can be a maximum number of primary amplified nucleic acids that can bind to a nucleic acid probe, for example, because of the maximum number of secondary amplified nucleic acids that can bind to a limited number of primary amplified nucleic acids and / or because of the maximum number of primary amplified nucleic acids that can bind to a limited number of read sequences on the nucleic acid probe. Although each potential position does not need to be actually filled with a signaling entity, this structure suggests that there is a saturation limit for the signaling entity, beyond which any other signaling entity that may happen to exist cannot associate with the nucleic acid probe or its target.

[0133] Therefore, certain embodiments of the present invention generally relate to systems and methods for amplifying a signal indicating a nucleic acid probe or its target, wherein the signal is saturable, i.e., such that there is a saturation upper limit to the number of signaling entities that can be associated with the nucleic acid probe or its target. Typically, the number is greater than 1. For example, the upper limit of the signaling entity can be at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 400, at least 500, etc. In some cases, the upper limit can be less than 500, less than 400, less than 300, less than 250, less than 200, less than 175, less than 150, less than 125, less than 100, less than 75, less than 50, less than 40, less than 30, less than 25, less than 20, less than 15, less than 10, less than 5, etc. In some cases, the upper limit can be determined as the maximum number of signaling entities that can be combined with secondary amplification nucleic acid, multiplied by the maximum number of secondary amplification nucleic acids that can be combined with primary amplification nucleic acids, multiplied by the maximum number of primary amplification nucleic acids that can be combined with the nucleic acid probe combined with the target. On the contrary, the technology such as rolling circle amplification or hairpin unfolding (hairpin unfolding) allows to amplify signals in an uncontrolled manner, that is, when there are enough reagents, amplification can proceed, without predetermined endpoint or saturation limit. Therefore, such technology does not have a theoretical upper limit for the number of signaling entities that can be associated with nucleic acid probe or its target.

[0134] However, it should be understood that the average number of signaling entities bound to a nucleic acid probe or its target is not necessarily the same as its upper limit, i.e., the signaling entities may not actually be at full saturation (although they may be fully saturated). For example, the saturation amount (or the number of signaling entities bound, relative to the maximum number that can be bound) may be less than 97%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, etc., and / or at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, etc. In some cases, allowing more time for binding to occur and / or increasing the concentration of the reagent can increase the saturation amount.

[0135] Because there is a potential upper limit to the number of signaling entities that can actually bind to a nucleic acid probe or its target, binding events distributed within a sample, e.g., spatially distributed, can exhibit substantially uniform size and / or brightness, such as those discussed above, as opposed to uncontrolled amplification. For example, because of the specific number of secondary amplified nucleic acids that can bind to primary amplified nucleic acids, it is found that the secondary amplified nucleic acids cannot exceed a fixed distance from the nucleic acid probe or its target, which can limit the "spot size" or diameter of fluorescence (indicative of binding) from the signaling entity.

[0136] In certain embodiments, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the binding events may exhibit substantially the same brightness, size (e.g., apparent diameter), color, etc., which may make it easier to distinguish the binding events from other events, such as nonspecific binding, etc.

[0137] In addition, as previously mentioned, some aspects of the present invention use code spaces encoding various binding events, and error detection and / or error correction can optionally be used to determine the combination of nucleic acid probes with their targets. In some cases, a group of nucleic acid probes can include certain "reading sequences" that can be combined with certain amplified nucleic acids, as discussed herein, and the position of the nucleic acid probe or target can be determined in a sample using a signaling entity associated with the amplified nucleic acid, for example, in certain code spaces, for example, as discussed herein. Also referring to International Patent Application Publication Nos. WO2016 / 018960 and WO 2016 / 018963, the entire contents of which are each incorporated herein by reference. As mentioned, in some cases, a group of reading sequences in the nucleic acid probe can be combined in various combinations, for example, so that a relatively large number of different nucleic acid probes can be determined using a relatively small number of reading sequences, as discussed herein.

[0138] Therefore, in some cases, a group of nucleic acid probes can each include a specific number of read sequences, some of which are shared between different nucleic acid probes, so that the nucleic acid probes of the whole group can include a specific number of read sequences. A group of nucleic acid probes can have any suitable number of read sequences. For example, a group of nucleic acid probes can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. Read sequences. In some embodiments, more than 20 are also possible. In addition, in some cases, a set of nucleic acid probes can have a total of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 20 or more, 24 or more, 32 or more, 40 or more, 50 or more, 60 or more, 64 or more, 100 or more, 128 or more, etc. possible reading sequences, although some or all of the probes may each contain more than one reading sequence, as discussed herein. In addition, in some embodiments, a set of nucleic acid probes can have no more than 100, no more than 80, no more than 64, no more than 60, no more than 50, no more than 40, no more than 32, no more than 24, no more than 20, no more than 16, no more than 15, no more than 14, no more than 13, no more than 12, no more than 11, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, or no more than 2 read sequences present. Combinations of any of these are also possible, for example, a set of nucleic acid probes can include a total of 10-15 read sequences.

[0139] As a non-limiting example of a way to combinatorially identify a relatively large number of nucleic acid probes from a relatively small number of read sequences contained within a nucleic acid probe, in a set of 6 different types of nucleic acid probes, each type of nucleic acid probe contains one or more read sequences, the total number of read sequences in the set may be no greater than 4. It should be understood that although 4 read sequences are used in this example for ease of explanation, in other embodiments, a larger number of nucleic acid probes can be implemented, for example, using 5, 8, 10, 16, 32, etc. or more read sequences, or any other suitable number of read sequences described herein, depending on the application. For example, if each nucleic acid probe contains two different read sequences, then by using 4 such read sequences (A, B, C, and D), up to 6 probes can be individually identified. It should be noted that in this example, the ordering of the read sequences on the nucleic acid probes is not critical, that is, "AB" and "BA" can be considered synonymous (although in other embodiments, the ordering of the read sequences can be critical, and "AB" and "BA" may not necessarily be synonymous). Similarly, if 5 read sequences (A, B, C, D, and E) are used in a set of nucleic acid probes, up to 10 probes (e.g., AB, AC, AD, AE, BC, BD, BE, CD, CE, DE) can be individually identified. For example, one of ordinary skill in the art will appreciate that for k read sequences in a set of n read sequences on each probe, up to 10 probes can be individually identified. Different probes are provided, assuming that the order of the read sequences is not important; because not all probe sequences need to have the same number of read sequences, and not all combinations of read sequences are necessarily used in each embodiment, different probes greater or less than this number may also be used in certain embodiments. In addition, it should also be understood that in some embodiments, the number of read sequences on each probe is not necessarily the same. For example, some probes may include 2 read sequences, while other probes may include 3 read sequences.

[0140] In some aspects, the binding pattern of the read sequence and / or nucleic acid probe within the sample can be used to define an error detection code and / or an error correction code, for example, to reduce or prevent the misidentification or error of nucleic acid. Therefore, for example, if the indication is combined (for example, as determined using a signaling entity), the position can be identified with "1"; on the contrary, if the indication is not combined, the position can be identified with "0" (or in some cases, vice versa). Then, multiple rounds of binding assays (for example, using different nucleic acid probes) can be used to establish a "code word", for example, for this spatial position. In some embodiments, the code word can be subject to error detection and / or correction. For example, the code word can be organized and configured so that for the binding pattern of the read sequence of a specified group or a specified nucleic acid probe, if no match is found, the match can be identified as an error, and, optionally, error correction can be applied to the sequence to determine the correct target for the nucleic acid probe. In some cases, the code word can have fewer "letters" or positions, that is, the total number of nucleic acids encoded by the code word, such as where each code word encodes different nucleic acids.

[0141] Such error detection and / or error correction codes can take various forms. Various such codes have been developed before in other fields such as the telecommunications industry, such as Golay codes or Hamming codes. In one group of embodiments, the binding patterns of the read sequences or nucleic acid probes are assigned so that not every possible combination is assigned.

[0142] For example, if 4 read sequences are possible and the nucleic acid probes contain 2 read sequences, then up to 6 nucleic acid probes can be identified; however, the number of nucleic acid probes used can be less than 6. Similarly, for k read sequences in a group with n read sequences on each nucleic acid probe, different probes, but the number of nucleic acid probes used can be greater or less than Additionally, these may be randomly assigned, or assigned in a specific manner to increase the ability to detect and / or correct errors.

[0143] As another example, if multiple rounds of nucleic acid probes are used, the rounds can be chosen arbitrarily. If in each round, each target can have two possible outcomes, such as being detected or not detected, there may be 2 n different targets, but the number of targets actually used can be less than 2 n For example, if in each round each target can have more than two possible outcomes, such as being detected in different color channels, then for n rounds there may be more than 2 n (For example, 3 n , 4 n, ...) different targets. In some cases, the number of targets actually used can be any number less than this number. In addition, these can be randomly assigned, or assigned in a specific way to increase the ability to detect and / or correct errors.

[0144] The codeword can be used to define various code spaces. For example, in one group of embodiments, the codeword or nucleic acid probe can be allocated in the code space so that the allocation interval is Hamming distance, and the number of wrong "readings" is measured in a specified mode, and the wrong "readings" cause the nucleic acid probe to be misinterpreted as different valid nucleic acid probes. In some cases, the Hamming distance can be at least 2, at least 3, at least 4, at least 5, at least 6, etc. In addition, in one group of embodiments, the allocation can be formed as a Hamming code, for example, Hamming (7, 4) code, Hamming (15, 11) code, Hamming (31, 26) code, Hamming (63, 57) code, Hamming (127, 120) code, etc. In another set of embodiments, the allocation may form a SECDED code, such as a SECDED (8, 4) code, a SECDED (16, 4) code, a SCEDED (16, 11) code, a SCEDED (22, 16) code, a SCEDED (39, 32) code, a SCEDED (72, 64) code, etc. In yet another set of embodiments, the allocation may form an extended binary Golay code, an optimal binary Golay code, or a ternary Golay code. In another set of embodiments, the allocation may represent a subset of possible values ​​obtained by any of the codes described above.

[0145] For example, an error detection code may be formed by limiting the number of codewords used to less than 10%, less than 5%, less than 2%, less than 1%, less than 0.1%, less than 0.01%, less than 0.001% of the total number of possible codewords, making it unlikely that an incorrect codeword exists as another used codeword. Thus, any detected codeword that does not match a used codeword is more likely to be incorrect.

[0146] For example, an error correction code can be formed by using only binary words that contain a fixed or constant number of "1" bits (or "0" bits) to encode the target. For example, the code space can include only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, etc. "1" bits (or "0" bits), for example, all codes have the same number of "1" bits or "0" bits, etc. In another set of embodiments, the allocation can represent a subset of possible values ​​obtained by the code described above to solve the problem of asymmetric readout errors. For example, in some cases, a code in which the number of "1" bits can be fixed for all binary code words used can eliminate measurement biases for code words with different numbers of "1" when the ratios of measuring a "0" bit as "1" or measuring a "1" bit as "0" are different.

[0147] Therefore, in some embodiments, once a codeword is determined (e.g., as discussed herein), the codeword can be compared to a known nucleic acid codeword. If a match is found, the nucleic acid target can be identified or determined. If a match is not found, the error in reading the codeword can be identified. In some cases, error correction can also be applied to determine the correct codeword, so the correct identity of the nucleic acid target is obtained. In some cases, the codeword can be selected so that assuming that there is only one error, only one possible correct codeword can be obtained, so the nucleic acid target may have only one correct identity. In some cases, this can also be extended to a larger codeword space or Hamming distance; for example, a codeword can be selected so that if there are 2, 3 or 4 errors (or more errors in some cases), only one possible correct codeword can be obtained, so the nucleic acid target may have only one correct identity.

[0148] The error correction code may be a binary error correction code, or it may be based on other numbering systems, such as ternary or quaternary error correction codes. For example, in one set of embodiments, more than one type of signaling entity may be used and assigned to different numbers within the error correction code. Thus, as a non-limiting example, a first signaling entity (or in some cases more than one signaling entity) may be assigned to "1", a second signaling entity (or in some cases more than one signaling entity) may be assigned to "2" ("0" indicates that no signaling entity exists), and the codeword is assigned to define a ternary error correction code. Similarly, a third signaling entity may be additionally assigned to "3" to form a ternary error correction code, and so on. Non-limiting examples of such codes include Reed-Solomon erasure codes and generalized forms thereof.

[0149] In addition, in some embodiments, the code can also be selected by the random selection of the subset of all possible code words.For example, the random subset of the binary code word of n codes can be selected to be of length.In some cases, these code words can be spaced with Hamming distance, that is, must be flipped so that one is converted into another so that some code words used keep the number of positions of some error robustness or error correction capability.In some embodiments, the random subset of the code words used can be measured using a mode such as next generation sequencing and error robustness and error correction can be selectively applied to the code words that meet the constraints required by these characteristics.

[0150] As discussed herein, in some aspects, a signaling entity is determined, for example, by imaging, to determine a nucleic acid probe and / or to form a codeword. Examples of signaling entities include those discussed herein. In some cases, a signaling entity within a sample can be, for example, spatially, measured using a variety of techniques. In some embodiments, the signaling entity can be fluorescent, and techniques for measuring fluorescence within a sample, such as fluorescence microscopy or confocal microscopy, can be used to spatially identify the position of the signaling entity within the cell. In some cases, the position of the entity within the sample can be determined in two dimensions or even three dimensions. In addition, in some embodiments, more than one signaling entity can be measured at one time (e.g., with signaling entities of different colors or emissions), and / or measured successively.

[0151] In addition, in some embodiments, the confidence level of a target (e.g., a nucleic acid target) can be determined. For example, the confidence level can be determined using the ratio of the number of exact matches to the number of matches with one or more one-bit errors. In some cases, the matching with only a confidence ratio greater than a specific value can be used. For example, in certain embodiments, only the confidence ratio of matching is greater than about 0.01, greater than about 0.03, greater than about 0.05, greater than about 0.1, greater than about 0.3, greater than about 0.5, greater than about 1, greater than about 3, greater than about 5, greater than about 10, greater than about 30, greater than about 50, greater than about 100, greater than about 300, greater than about 500, greater than about 1000 or any other suitable value, and the matching is acceptable. Additionally, in some embodiments, a match is accepted only if the confidence ratio of the target is greater than about 0.01, about 0.03, about 0.05, about 0.1, about 0.3, about 0.5, about 1, about 3, about 5, about 10, about 30, about 50, about 100, about 300, about 500, about 1000, or any other suitable value, relative to the internal standard or false positive control.

[0152] In some embodiments, the spatial position of an entity (and therefore, a nucleic acid probe that can be associated with the entity) can be determined with a relatively high resolution. For example, the position can be determined with a spatial resolution of greater than about 100 microns, greater than about 30 microns, greater than about 10 microns, greater than about 3 microns, greater than about 1 micron, greater than about 800 nm, greater than about 600 nm, greater than about 500 nm, greater than about 400 nm, greater than about 300 nm, greater than about 200 nm, greater than about 100 nm, greater than about 90 nm, greater than about 80 nm, greater than about 70 nm, greater than about 60 nm, greater than about 50 nm, greater than about 40 nm, greater than about 30 nm, greater than about 20 nm, or greater than about 10 nm.

[0153] There are various techniques that can optically determine the spatial position of an entity or image the spatial position of an entity, for example, using fluorescence microscopy. In some embodiments, more than one color can be used. In some cases, the spatial position can be determined with super-resolution, or with a resolution higher than the wavelength of light or the diffraction limit. Non-limiting examples include STORM (random optical reconstruction microscopy), STED (stimulated emission depletion microscopy), NSOM (near-field scanning optical microscopy), 4Pi microscopy, SIM (structured illumination microscopy), SMI (spatial modulated light illumination) microscopy, RESOLFT (reversible saturated optical linear fluorescence transition microscopy), GSD (ground state loss microscopy), SSIM (saturated structured illumination microscopy), SPDM (spectral precision distance microscopy), photoactivated localization microscopy (PALM), fluorescence photoactivated localization microscopy (FPALM), LIMON (3D light microscopy nanomicroscopy), super-resolution optical wave imaging (SOFI), etc. See, for example, U.S. Pat. No. 7,838,302 to Zhuang et al., issued on November 23, 2010, entitled “Sub-Diffraction Limit Image Resolution and Other Imaging Techniques”; U.S. Pat. No. 8,564,792 to Zhuang et al., issued on October 22, 2013, entitled “Sub-diffraction Limit Image Resolution in Three Dimensions”; or International Patent Application Publication No. WO 2013 / 090360 to Zhuang et al., published on June 20, 2013, entitled “High Resolution Dual-Objective Microscopy”, the entire contents of each of these patents are incorporated herein by reference.

[0154] As an illustrative non-limiting example, in one set of embodiments, the sample can be imaged using a high numerical aperture, oil immersion objective with a 100X magnification and light collected on an electron multiplying CCD camera. In another embodiment, the sample can be imaged using a high numerical aperture, oil immersion lens with a 40X magnification and light collected using a wide-field scientific grade CMOS camera. Using different combinations of objective lenses and cameras, in various non-limiting embodiments, a single field of view can correspond to no less than 40x 40 microns, 80x 80 microns, 120x 120 microns, 240x 240 microns, 340x 340 microns, or 500x 500 microns, etc. Similarly, in some embodiments, a single camera pixel can correspond to an area of ​​no less than 80x80 nm, 120x120 nm, 160x160 nm, 240x240 nm, or 300x300 nm, etc. of the sample. In another embodiment, the sample can be imaged using a low numerical aperture, air lens with 10X magnification and light collected using a sCMOS camera. In other embodiments, the sample can be optically sectioned by illumination with a single or multiple scanning diffraction limited focus, which is generated by a scanning mirror or a rotating disk and collected through a single or multiple pinholes. In another embodiment, the sample can also be illuminated by a thin sheet of light, which is generated by any of a variety of methods known to those skilled in the art.

[0155] In one embodiment, the sample can be illuminated by a single Gaussian mode laser line. In some embodiments, the illumination spectrum can be planarized by passing these laser lines through a multimode optical fiber that is vibrated by a piezoelectric or other mechanical device. In some embodiments, the illumination spectrum can be planarized by passing a single mode Gaussian beam through a variety of refractive beam shapers, such as a piShaper or a series of stacked Powell prisms. In yet another set of embodiments, the Gaussian beam can be passed through a variety of different diffusing elements, such as frosted glass or engineered diffusers, which in some cases can be rotated at high speed to remove residual laser speckle. In yet another embodiment, the laser illumination light can be passed through a series of microlens arrays to produce overlapping images of the illumination light that approximate a planar illumination field.

[0156] In some embodiments, the centroid of the spatial location of the entity can be determined. For example, the centroid of the signaling entity can be determined within an image or a series of images using an image analysis algorithm known to those of ordinary skill in the art. In some cases, the algorithm can be selected to determine non-overlapping single emitters and / or partially overlapping single emitters in a sample. Non-limiting examples of suitable techniques include maximum likelihood algorithms, least squares algorithms, Bayesian algorithms, compressed sensing algorithms, and the like. In some cases, combinations of these techniques can also be used.

[0157] In addition, in some cases, the signaling entity can be inactivated. For example, in some embodiments, a first secondary nucleic acid probe that can associate with a signaling entity (e.g., using an amplified nucleic acid) can be applied to a sample, the first secondary nucleic acid probe can recognize a first read sequence (e.g., on the nucleic acid probe), and then the signaling entity can be inactivated before a second secondary nucleic acid probe is applied to the sample, for example, the second secondary nucleic acid probe can associate with the signaling entity (e.g., using an amplified nucleic acid). If multiple signaling entities are used, the same or different techniques can be used to inactivate the signaling entity, and some or all of the multiple signaling entities can be inactivated, for example, sequentially or simultaneously.

[0158] Inactivation can be caused by removing the signaling entity (e.g., from a sample or from a nucleic acid probe, etc.), and / or by chemically altering the signaling entity in some manner (e.g., by photobleaching the signaling entity, bleaching or chemically altering the structure of the signaling entity, e.g., by reduction, etc.). For example, in one set of embodiments, the fluorescent signaling entity can be inactivated by chemical or optical techniques such as oxidation, photobleaching, chemical bleaching, stringent washing or enzymatic digestion or by reaction by exposure to an enzyme, dissociation of the signaling entity from other components (e.g., probes), chemical reaction of the signaling entity (e.g., chemical reaction with a reactant that can alter the structure of the signaling entity), etc. For example, bleaching can occur by exposure to oxygen, a reducing agent, or the signaling entity can be chemically cleaved from the nucleic acid probe (e.g., using tris(2-carboxyethyl)phosphine) and washed away by a fluid stream.

[0159] In some embodiments, various nucleic acid probes (including primary and / or secondary nucleic acid probes) can be associated with one or more signaling entities, for example, using amplified nucleic acids, as discussed herein. If more than one nucleic acid probe is used, each of the signaling entities can be the same or different. In certain embodiments, a signaling entity is any entity capable of emitting light. For example, in one embodiment, the signaling entity is fluorescent. In other embodiments, the signaling entity can be phosphorescent, radioactive, absorptive, etc. In some cases, the signaling entity is any entity that can be determined within a sample at a relatively high resolution, for example, at a resolution higher than the wavelength or diffraction limit of visible light. The signaling entity can be, for example, a dye, a small molecule, a peptide or a protein, etc. In some cases, the signaling entity can be a single molecule. If multiple secondary nucleic acid probes are used, the nucleic acid probe can be associated with the same or different signaling entities or contain the same or different signaling entities.

[0160] Non-limiting examples of signaling entities include fluorescent entities (fluorophores) or phosphorescent entities, for example, cyanine dyes (e.g., Cy2, Cy3, Cy3B, Cy5, Cy5.5, Cy7, etc.), Alexa Fluor dyes, Atto dyes, photoconvertible dyes, photoactivated dyes, fluorescent dyes, metal nanoparticles, semiconductor nanoparticles or "quantum dots", fluorescent proteins such as GFP (green fluorescent protein), or photoactivated fluorescent proteins such as PAGFP, PSCFP, PSCFP2, Dendra, Dendra2, EosFP, tdEos, mEos2, mEos3, PAmCherry, PAtagRFP, mMaple, mMaple2, and mMaple3. Other suitable signaling entities are known to those of ordinary skill in the art. See, for example, U.S. Patent No. 7,838,302 or International Patent Application Publication No. WO 2015 / 160690, the entire contents of each of which are incorporated herein by reference.

[0161] In one set of embodiments, the signaling entity may be attached to the oligonucleotide sequence via a bond that may be cleaved to release the signaling entity. In one set of embodiments, the fluorophore may be conjugated to the oligonucleotide via a cleavable bond, such as a photocleavable bond. Non-limiting examples of photocleavable bonds include, but are not limited to, 1-(2-nitrophenyl)ethyl, 2-nitrobenzyl, biotin phosphoramidite, acrylic acid phosphoramidite, diethylaminocoumarin, 1-(4,5-dimethoxy-2-nitrophenyl)ethyl, cyclododecyl(dimethoxy-2-nitrophenyl)ethyl, 4-aminomethyl-3-nitrobenzyl, (4-nitro-3-(1-chlorocarbonyloxyethyl)phenyl)methyl-S-acetylthioate, (4-nitro-3-(1-chlorocarbonyloxyethyl)phenyl)methyl-3-(2-pyridyldithiopropionate), 3-(4,4'-dimethoxytrimethyl)-1-(2-nitrophenyl)-propane-1,3-dithioate ]-phosphoramidite, 1-[2-nitro-5-(6-(4,4'-dimethoxytrimethyloxy)butyramidomethyl)phenyl]-ethyl-[2-cyanoethyl-(N,N-diisopropyl)]-phosphoramidite, 1-[2-nitro-5-(6-(4,4'-dimethoxytrimethyloxy)butyramidomethyl)phenyl]-ethyl-[2-cyanoethyl-(N,N-diisopropyl)]-phosphoramidite, 1-[2-nitro-5-(6-(N-(4,4'-dimethoxytrimethyloxy)butyramidomethyl)phenyl]-ethyl-[2-cyanoethyl-(N,N-diisopropyl)]-phosphoramidite, 1-[2-nitro-5-(6-(N-(4,4'-dimethoxytrimethyl))-biotinamidohexanoyl-methyl)phenyl]-ethyl-[2-cyanoethyl-(N,N-diisopropyl)]-phosphoramidite, or similar linkers. The oligonucleotide sequence can be, for example, a primary or secondary (or other) amplification nucleic acid, such as those discussed herein.

[0162] In another set of embodiments, the fluorophore can be conjugated to the oligonucleotide via a disulfide bond. The disulfide bond can be cleaved by a variety of reducing agents, such as, but not limited to, dithiothreitol, dithioerythritol, β-mercaptoethanol, sodium borohydride, thioredoxin, glutaredoxin, trypsinogen, hydrazine, diisobutylaluminum hydride, oxalic acid, formic acid, ascorbic acid, phosphorous acid, tin chloride, glutathione, thioglycolate, 2,3-dimercaptopropanol, 2-mercaptoethylamine, 2-aminoethanol, tris(2-carboxyethyl)phosphine, di(2-mercaptoethyl)sulfone, N,N'-dimethyl-N,N'-di(mercaptoacetyl)hydrazine, 3-mercaptopropionate, dimethylformamide, thiopropyl-agarose, tri-n-butylphosphine, cysteine, ferrous sulfate, sodium sulfite, phosphite, hypophosphite, phosphorothioate, etc., and / or any combination of these. The oligonucleotide sequence can be, for example, a primary or secondary (or other) amplification nucleic acid, such as those discussed herein.

[0163] In another embodiment, the fluorophore can be conjugated to the oligonucleotide by one or more thiophosphate-modified nucleotides (wherein the sulfur modification replaces the bridge oxygen and / or non-bridge oxygen). In certain embodiments, the fluorophore can be cleaved from the oligonucleotide by adding a compound, such as but not limited to iodoethanol, iodine, silver nitrate, or mercuric chloride mixed in ethanol. In another set of embodiments, the signaling entity can be chemically inactivated by reduction or oxidation. For example, in one embodiment, chromophores such as Cy5 or Cy7 can be reduced to a stable non-fluorescent state using sodium borohydride. In another set of embodiments, the fluorophore can be conjugated to the oligonucleotide by an azo bond, and the azo bond can be cleaved using 2-[(2-N-arylamino)phenylazo]pyridine. In another set of embodiments, the fluorophore can be conjugated to the oligonucleotide by a suitable nucleic acid segment, and the nucleic acid segment can be cleaved when appropriately exposed to a DNA enzyme, such as an exodeoxyribonuclease or an endodeoxyribonuclease. Examples include, but are not limited to, deoxyribonuclease I or deoxyribonuclease II. In one group of embodiments, the cleavage can occur by restriction endonucleases. Non-limiting examples of potential suitable restriction endonucleases include BamHI, BsrI, NotI, XmaI, PspAI, DpnI, MboI, MnlI, Eco57I, Ksp632I, DraIII, AhaII, SmaI, MluI, HpaI, ApaI, BclI, BstEII, TaqI, EcoRI, SacI, HindII, HaeII, DraII, Tsp509I, Sau3AI, PacI, etc. More than 3000 restriction enzymes have been studied in detail, and more than 600 of these are commercially available. In another group of embodiments, the fluorophore can be conjugated to biotin, and the oligonucleotide is conjugated to avidin or streptavidin. The interaction between biotin and avidin or streptavidin allows the fluorophore to be conjugated to the oligonucleotide, and when fully exposed to excess additives, the free biotin can "compete for" the connection, thereby causing cleavage to occur. In addition, in another set of embodiments, the probe can be removed using a corresponding "toe-hold-probe", which contains the same sequence as the probe, and an additional number of bases homologous to the encoded probe (e.g., 1-20 additional bases, e.g., 5 additional bases). These probes can remove the labeled readout probes through strand displacement interactions. The oligonucleotide sequence can be, for example, a primary or secondary (or other) amplification nucleic acid, such as those discussed herein.

[0164] As used herein, the term "light" generally refers to electromagnetic radiation having any suitable wavelength (or equivalently, frequency). For example, in some embodiments, light can include wavelengths in the optical or visible range (e.g., having a wavelength of about 400nm-about 700nm, i.e., "visible light"), infrared wavelengths (e.g., having a wavelength of about 300 microns-700nm), ultraviolet wavelengths (e.g., having a wavelength of about 400nm-about 10nm), etc. In some cases, as discussed in detail below, more than one entity can be used, i.e., chemically different or unique entities, e.g., structurally different or unique entities. However, in other cases, the entities can be chemically identical or at least substantially chemically identical.

[0165] In one set of embodiments, the signaling entity is "convertible", that is, the entity can be converted between two or more states, at least one of which emits light of a desired wavelength. In one or more other states, the entity may not emit light, or emit light of a different wavelength. For example, an entity can be "activated" to a first state capable of producing light of a desired wavelength, and "deactivated" to a second state incapable of emitting light of the same wavelength. If an entity can be activated by incident light of a suitable wavelength, the entity is "photoactivatable". As a non-limiting example, Cy5 can be converted between fluorescent and dark states by light of different wavelengths in a controlled and reversible manner, that is, 633nm (or 642nm, 647nm, 656nm) red light can convert or inactivate Cy5 to a stable dark state, while 405nm green light can convert or activate Cy5 back to a fluorescent state. In some cases, the entity can be reversibly converted between two or more states, for example, when exposed to a suitable stimulus. For example, a first stimulus (e.g., light of a first wavelength) can be used to activate a convertible entity, and a second stimulus (e.g., light of a second wavelength) can be used to deactivate the convertible entity, e.g., to a non-emitting state. Any suitable method can be used to activate the entity. For example, in one embodiment, incident light of a suitable wavelength can be used to activate the entity to emit light, i.e., the entity is "photoconvertible". Therefore, the photoconvertible entity can be converted between different light emitting or non-emitting states by, for example, incident light of different wavelengths. The light can be monochromatic (e.g., generated using a laser) or polychromatic. In another embodiment, the entity can be activated after being stimulated by an electric field and / or a magnetic field. In other embodiments, the entity can be activated after being exposed to a suitable chemical environment, e.g., by adjusting pH, or inducing a reversible chemical reaction involving the entity, etc. Similarly, any suitable method can be used to deactivate the entity, and the methods for activating and deactivating the entity do not have to be the same. For example, the entity can be deactivated after being exposed to incident light of a suitable wavelength, or the entity can be deactivated by being placed for a sufficient time.

[0166] Typically, a "switchable" entity can be identified by a person of ordinary skill in the art by determining conditions under which the entity in the first state can emit light when exposed to an excitation wavelength, converting the entity from a first state to a second state, for example, after exposure to light of a switching wavelength, and then showing that the entity can no longer emit light (or emits light of much reduced intensity) when exposed to an excitation wavelength while in the second state.

[0167] In one set of embodiments, as discussed, the switchable entity can be switched upon exposure to light. In some cases, the light used to activate the switchable entity can come from an external source, for example, a light source such as a laser light source, another light emitting entity in close proximity to the switchable entity, etc. In some cases, the second light emitting entity can be a fluorescent entity, and in certain embodiments, the second light emitting entity itself can also be a switchable entity.

[0168] In some embodiments, the switchable entity includes a first light emitting portion (e.g., a fluorophore), and a second portion that activates or "converts" the first portion. For example, upon exposure to light, the second portion of the switchable entity can activate the first portion, causing the first portion to emit light. Examples of activator portions include, but are not limited to, Alexa Fluor 405 (Invitrogen), Alexa Fluor 488 (Invitrogen), Cy2 (GE Healthcare), Cy3 (GE Healthcare), Cy3B (GE Healthcare), Cy3.5 (GE Healthcare), or other suitable dyes. Examples of light emitting moieties include, but are not limited to, Cy5, Cy5.5 (GE Healthcare), Cy7 (GE Healthcare), AlexaFluor 647 (Invitrogen), Alexa Fluor 680 (Invitrogen), Alexa Fluor 700 (Invitrogen), Alexa Fluor 750 (Invitrogen), Alexa Fluor 790 (Invitrogen), DiD, DiR, YOYO-3 (Invitrogen), YO-PRO-3 (Invitrogen), TOT-3 (Invitrogen), TO-PRO-3 (Invitrogen), or other suitable dyes.These can be linked together, e.g., covalently, e.g., directly, or through a linker, e.g., to form compounds such as, but not limited to, Cy5-Alexa Fluor 405, Cy5-Alexa Fluor 488, Cy5-Cy2, Cy5-Cy3, Cy5-Cy3.5, Cy5.5-Alexa Fluor 405, Cy5.5-Alexa Fluor 488, Cy5.5-Cy2, Cy5.5-Cy3, Cy5.5-Cy3.5, Cy7-Alexa Fluor 405, Cy7-Alexa Fluor 488, Cy7-Cy2, Cy7-Cy3, Cy7-Cy3.5, Alexa Fluor 647-Alexa Fluor 405, Alexa Fluor 647-Alexa Fluor 488, Alexa Fluor 647-Cy2, Alexa Fluor In some embodiments, the activator moiety can be substituted with Alexa Fluor 647-Cy3, Alexa Fluor 647-Cy3.5, Alexa Fluor 750-Alexa Fluor 405, Alexa Fluor 750-Alexa Fluor 488, AlexaFluor 750-Cy2, Alexa Fluor 750-Cy3, or Alexa Fluor 750-Cy3.5. Those of ordinary skill in the art know the structures of these and other compounds, many of which are commercially available. The moiety can be connected by a covalent bond, or connected by a joint, such as those described in detail below. Other light emitting or activator moieties can include a moiety with two quaternary ammonium nitrogen atoms connected by a polymethylene chain, wherein each nitrogen is independently a part of a heteroaromatic moiety, such as pyrrole, imidazole, thiazole, pyridine, quinoline, indole, benzothiazole, etc., or a part of a non-aromatic amine. In some cases, 5, 6, 7, 8, 9 or more carbon atoms can be present between two nitrogen atoms.

[0169] In some cases, the light emitting portion and the activator portion, when separated from each other, can each be a fluorophore, that is, an entity that can emit light of a specific emission wavelength when exposed to a stimulus (e.g., an excitation wavelength). However, when forming a convertible entity comprising a first fluorophore and a second fluorophore, the first fluorophore forms a first light emitting portion, and the second fluorophore forms an activator portion, which activates or "converts" the first portion in response to the stimulus. For example, the convertible entity can include a first fluorophore that is directly bonded to a second fluorophore, or the first or second entity can be connected by a joint or a common entity. Whether a pair of light emitting portions and activator portions produce a suitable convertible entity can be tested by methods known to those of ordinary skill in the art. For example, light of various wavelengths can be used to stimulate the pair, and the emitted light from the light emitting portion can be measured to determine whether the pair forms a suitable conversion.

[0170] As a non-limiting example, Cy3 and Cy5 can be linked together to form such an entity. In this example, Cy3 is an activator part that can activate Cy5 (light emitting part). Therefore, light that is at or near the activation or maximum absorption of the second part of the entity (e.g., for Cy3, close to 532nm light) can enable the part to activate the first light emitting part, thereby enabling the first part to emit light (e.g., for Cy5, close to 647nm). See, for example, U.S. Patent No. 7,838,302, the entire contents of which are incorporated herein by reference. In some cases, the first light emitting part can be subsequently inactivated by any suitable technology (e.g., by directing 647nm red light to the Cy5 part of the molecule).

[0171] Other non-limiting examples of potentially suitable activator moieties include 1,5IAEDANS, 1,8-ANS, 4-methylumbelliferone, 5-carboxy-2,7-dichlorofluorescein, 5-carboxyfluorescein (5-FAM), 5-carboxynaphthofluorescein, 5-carboxytetramethylrhodamine (5-TAMRA), 5-FAM (5-carboxyfluorescein), 5-HAT (hydroxytryptamine), 5-hydroxytryptamine (HAT), 5-ROX (carboxy-X-rhodamine), 5-TAMRA (5-carboxytetramethylrhodamine), 6-carboxyrhodamine 6G, 6-CR 6G, 6-JOE, 7-amino-4-methylcoumarin, 7-aminoactinomycin D (7-AAD), 7-hydroxy-4-methylcoumarin, 9-amino-6-chloro-2-methoxyacridine, ABQ, acid fuchsin, ACMA (9-amino-6-chloro-2-methoxyacridine), acridine orange, acridine red, acridine yellow, acriflavine, acriflavine Feulgen SITSA, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor488, 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 Fluor633, Alexa Fluor 635, Alizarin aminocarboxylic acid complex, Alizarin red, AMC, AMCA-S, AMCA (aminomethylcoumarin), AMCA-X, aminoactinomycin D, aminocoumarin, aminomethylcoumarin (AMCA), aniline blue, Anthrocylstearate, APTRA-BTC, APTS, Astrazon Brilliant Red 4G, Basic Orange R, Basic Red 6B, Basic Yellow 7GLL, Atabrine, ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO520, ATTO 532, ATTO 550, ATTO 565, ATTO 590, ATTO 594, ATTO 610, ATTO 611X, ATTO 620, ATTO 633, ATTO 635, ATTO 647, ATTO 647N, ATTO 655, ATTO 680, ATTO 700, ATTO 725, ATTO740, ATTO-TAG CBQCA, ATTO-TAG FQ, Auramine, Aurophosphine G, Aurophosphine,BAO 9 (diaminophenyl oxadiazole), BCECF (high pH), BCECF (low pH), berberine sulfate, Bimane, benzamide, benzhydrylamine (Hoechst), di-BTC, Blancophor FFG, Blancophor SV, BOBO-1, BOBO-3, Bodipy 492 / 515, Bodipy 493 / 503, Bodipy 500 / 510, Bodipy 505 / 515, Bodipy 530 / 550, Bodipy 542 / 563, Bodipy 558 / 568, Bodipy 564 / 570, Bodipy 576 / 589, Bodipy 581 / 591, Bodipy 630 / 650-X, Bodipy 650 / 665-X, Bodipy 665 / 676, Bodipy Fl, Bodipy FL ATP, Bodipy Fl-ceramide, Bodipy R6G, Bodipy TMR, Bodipy TMR-X conjugate, Bodipy TMR-X, SE, Bodipy TR, Bodipy TRATP, Bodipy TR-X SE, BO-PRO-1, BO-PRO-3, Brilliant Sulphoflavin FF, BTC, BTC-5N, Calcein, Calcein Blue, Calcium Crimson, Calcium Green, Calcium Green-1Ca, 2+ Dye, Calcium Green-2Ca 2+ , Calcium Green-5N Ca 2+ , Calcium Green-C18 Ca 2+ , Calcium Orange, Calcofluor White, Carboxy-X-Rhodamine (5-ROX), Cascade Blue, Cascade Yellow, Catecholamine, CCF2 (GeneBlazer), CFDA, Chromomycin A, Chromomycin A, CL-NERF, CMFDA, Coumarin Phalloidin, CPM Methyl Coumarin, CTC, CTC Methyl Cy2, Cy3.1 8, Cy3.5, Cy3, Cy5.1 8, Cyclic AMP Fluoro Sensor (FiCRhR), Dabcyl, Dansyl, Dansyl Amine, Dansyl Cadaverine, Dansyl Chloride, Dansyl DHPE, Dansyl Fluoride, DAPI, Dapoxyl, Dapoxyl 2, Dapoxyl3'DCFDA, DCFH (dichlorodihydrofluorescein diacetate), DDAO, DHR (dihydrorhodamine 123), di-4-ANEPPS, di-8-ANEPPS (non-ratio), DiA (4-Di-16-ASP), dichlorodihydrofluorescein diacetate (DCFH), DiD - lipophilic tracer, DiD (DiIC18 (5)), DIDS, dihydrorhodamine 123 (DHR), DiI (DiIC18 (3)), dinitrophenol, DiO (DiOC18 (3)), DiR, DiR (DiIC18 (7)), DM-NERF (high pH), DNP, dopamine, DTAF, DY-630-NHS, DY-635-NHS, DyLight 405, DyLight 488, DyLight 549, DyLight633, DyLight 649, DyLight 680, DyLight 800, ELF 97, Eosin, Erythrosin, Erythrosin ITC, Ethidium bromide, Ethidium bromide homodimer-1 (EthD-1), Acridine Orange, EukoLight, Europium (III) chloride, Fast Blue, FDA, Feulgen (pararosaniline), FIF (formaldehyde-induced fluorescence), FITC, Flazo Orange, Fluo-3, Fluo-4, Fluorescein (FITC), Fluorescein diacetate, Fluoro-Emerald, Fluoro-Gold (hydroxy Hydroxystilbamidine), Fluor-Ruby, FluorX, FM 1-43, FM 4-46, Fura Red (high pH), FuraRed / Fluo-3, Fura-2, Fura-2 / BCECF, Genacryl Brilliant Red B, Genacryl Brilliant Yellow 10GF, Genacryl Pink 3G, Genacryl Yellow 5GF, GeneBlazer (CCF2), Gloxalic Acid, Granular Blue, Hematoporphyrin, Hoechst 33258, Hoechst 33342, Hoechst 34580, HPTS, Hydroxycoumarin, Hydroxy FluoroGold, Serotonin, Indo-1, High Calcium, Indo-1, Low Calcium, Indole Dicarbocyanine (DiD), Indole Tricarbocyanine (DiR), Intrawhite Cf, JC-1, JO-JO-1, JO-PRO-1, LaserPro, Laurodan, LDS 751 (DNA), LDS 751 (RNA), Leucophor PAF, Leucophor SF, Leucophor WS, Lissamine Rhodamine, Lissamine Rhodamine B, Calcein / Ethifradiin Homodimer, LOLO-1, LO-PRO-1, Fluoro Yellow, Lyso Tracker Blue, LysoTracker Blue-White, Lyso Tracker Green, Lyso Tracker Red, Lyso Tracker Yellow, LysoSensor Blue, LysoSensor Green, LysoSensor Yellow / Blue, Mag Green, Magdala Red (root bark red B), Mag-Fura Red, Mag-Fura-2, Mag-Fura-5, Mag-Indo-1, Magnesium Green, Magnesium Orange, Malachite Green, Marina Blue, Maxilon Brilliant Flavin 10GFF, Maxilon Brilliant Flavin 8GFF, Merocyanine, Methoxycoumarin, Mitotracker Green FM, Mitotracker Orange, Mitotracker Red, Mithramycin, Monobromodiamine, Monobromodiamine (mBBr-GSH), Monochlorodiamine, MPS (Methyl Green Pyronitrile), NBD, NBD Amine, Nile Red, Nitrobenzoxadidole, Norepinephrine, Nuclear Fast Red, Nuclear Yellow, Nylosan Brilliant Iavin E8G, Oregon Green, Oregon Green 488-X, OregonGreen, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, Feulgen, PBFI, Sudan Red B, Phorwite AR, Phorwite BKL, Phorwite Rev, Phorwite RPA, Phosphine 3R, PKH26 (Sigma), PKH67, PMIA, Pontochrome Blue Black, POPO-1,POPO-3, PO-PRO-1, PO-PRO-3, Primulina, Reactive Yellow, Propidium Iodide (PI), PyMPO, Pyrene, Pyronine, Pyronine B, PyrozalBrilliant Flavin 7GF, QSY 7, Quinacridone, Resorufin, RH 414, Rhod-2, Rhodamine, Rhodamine 110, Rhodamine 123, Rhodamine 5GLD, Rhodamine 6G, Rhodamine B, Rhodamine B 200, Rhodamine B extra, Rhodamine BB, Rhodamine BG, Rhodamine Green, Rhodamine Phallicidine, Rhodamine Phalloidin, Rhodamine Red, Rhodamine WT, Rose Bengal, S65A, S65C, S65L, S65T, SBFI, 5-HT, Sevron Brilliant Red 2B, Sevron Brilliant Red 4G, Sevron Brilliant Red B, Sevron Orange, Sevron Yellow L, SITS, SITS (Primrose), SITS (Stilbene Isothiosulfonic Acid), SNAFL Calcein, SNAFL-1, SNAFL-2, SNARF Calcein, SNARF1, SodiumGreen, SpectrumAqua, SpectrumGreen, SpectrumOrange, Spectrum Red, SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium), Stilbene, Sulforhodamine B can C, Sulforhodamine Extra, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 15, SYTO 16, SYTO 17, SYTO 18, SYTO 20, SYTO 21, SYTO 22, SYTO 23, SYTO 24, SYTO 25, SYTO 40, SYTO 41, SYTO SYTO 42, SYTO 43, SYTO 44, SYTO 45, SYTO 59, SYTO60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO 80, SYTO 81, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTOX Blue, SYTOX Green, SYTOX Orange, Tetracycline, Tetramethylrhodamine (TAMRA), Texas Red, Texas Red-X conjugate, Thiadicarbocyanine (DiSC3), Thiazide Red R, Thiazide Orange, Thioflavin 5, Thioflavin S, Thioflavin TCN, Thiolyte, Thiolyte Orange, Tinopol CBS (fluorescent brightener), TMR, TO-PRO-1, TO-PRO-3, TO-PRO-5, TOTO-1, TOTO-3,TRITC (tetramethylrhodamine isothiocyanate), True Blue, TruRed, Ultralite, Uranine B, Uvitex SFC, WW 781, X-rhodamine, XRITC, Xylene Orange, Y66F, Y66H, Y66W, YO-PRO-1, YO-PRO-3, YOYO-1, YOYO-3, SYBR Green, Thiazole Orange (inter-chelating dye), or a combination thereof.

[0172] Another aspect of the present invention relates to computer-implemented methods. For example, a computer and / or automated system capable of automatically and / or repeatedly performing any of the methods described herein may be provided. As used herein, an "automated" device refers to a device that can operate without human guidance, that is, an automated device can perform a function during a period of time after anyone has completed any action to facilitate a function (e.g., by inputting instructions to a computer to start the process). Typically, the automated device can perform repeated functions on time after this point. In some cases, the processing steps may also be recorded on a machine-read-only medium.

[0173] For example, in some cases, a computer can be used to control the imaging of a sample, for example, using fluorescence microscopy, STORM or other super-resolution techniques such as those described herein. In some cases, the computer can also control operations such as drift correction, physical registration, hybridization and cluster alignment in image analysis, cluster decoding (e.g., fluorescence cluster decoding), error detection or correction (e.g., as described herein), noise reduction, identification of foreground features from background features (such as noise or debris in an image), etc. As an example, a computer can be used to control the activation and / or excitation of a signaling entity within a sample, and / or the acquisition of an image of a signaling entity. In one set of embodiments, a sample can be excited using light having various wavelengths and / or intensities, and the order of the wavelengths of light used to excite the sample can be associated with an image acquired from a sample containing the signaling entity using a computer. For example, a computer can apply light having various wavelengths and / or intensities to a sample so as to produce a different average number of signaling entities in each target area (e.g., one activated entity per position, two activated entities per position, etc.). In some cases, this information can be used to construct an image of the signaling entity and / or determine the location of the signaling entity, in some cases at high resolution, as described above.

[0174] In some aspects, the sample is placed on a microscope. In some cases, the microscope may include one or more channels, such as microfluidic channels, to guide or control fluid to flow to or from a sample. For example, in one embodiment, nucleic acid probes such as those discussed herein may be introduced into a sample and / or removed from a sample by allowing fluid to flow to or from a sample through one or more channels. In some cases, one or more chambers or reservoirs may also be present for retaining fluid, for example, in communication with a channel and / or with a sample fluid. Those of ordinary skill in the art are familiar with channels, including microfluidic channels, for moving fluid to or from a sample.

[0175] The following documents are each incorporated herein by reference in their entirety: U.S. Patent No. 10,240,146, entitled “Probe Library Construction”; U.S. Patent Application Publication No. 2017 / 0220733, entitled “Systems and Methods for Determining Nucleic Acids”; U.S. Patent Application Serial No. 62 / 779,333, entitled “Amplification Methods and Systems for MERFISH and Other Applications”; International Patent Application Publication No. WO 2016 / 018960, entitled “Systems and Methods for Determining Nucleic Acids”; International Patent Application Publication No. WO 2016 / 018963, entitled “Probe Library Construction”; International Patent Application Publication No. WO 2018 / 089445, entitled “Matrix imprinting and Clearing”; International Patent Application Publication No. WO 2018 / 218150, entitled “Systems and Methods for High-Throughput Image-Based and International Patent Application Publication No. WO 2018 / 089438, entitled “Multiplexed Imaging Using MERFISH and Expansion Microscopy”. In addition, the entire contents of each of the following are also incorporated herein by reference: U.S. Provisional Patent Application Serial No. 62 / 836,578, entitled “Imaging-Based Pooled CRISPR Screening” filed by Zhuang et al. on April 19, 2019 and U.S. Provisional Patent Application Serial No. 62 / 841,715, entitled “Imaging-Based Pooled CRISPR Screening” filed by Zhuang et al. on May 1, 2019.

[0176] The following examples are intended to illustrate certain embodiments of the invention, but are not intended to represent the full scope of the invention.

[0177] Example 1

[0178] Mixed library CRISPR screening provides a powerful means to discover genetic factors involved in cellular processes in a high-throughput manner. However, the phenotypes available for mixed library screening are limited. Complex phenotypes, such as cell morphology and subcellular molecular organization, and their dynamics, require imaging-based readouts, which are currently not available for mixed library CRISPR screening. These embodiments show all mixed library CRISPR screening methods based on imaging, which combine high-content phenotypic imaging in single cells with high-throughput guide RNA (sgRNA) identification. In one such method, sgRNA is co-delivered to cells, wherein the corresponding barcode is placed in the 3' untranslated region (3'UTR) of the reporter gene using a lentiviral delivery system, and the lentiviral delivery system has a reduced recombination-induced sgRNA-barcode mismatch. Multiple error robust fluorescence in situ hybridization (MERFISH) can be used to read out the barcode, so sgRNA can be identified with high accuracy. See, for example, International Patent Application Publication Nos. WO 2016 / 018960, WO2016 / 018963, WO 2018 / 089445, WO 2018 / 218150, WO2018 / 089438 and WO 2018 / 089438, the entire contents of each of which are incorporated herein by reference. These embodiments use this approach to screen 162 sgRNAs targeting 54 RNA-binding proteins for the effect of RNA localization to the nuclear compartment, and to find previously unknown regulatory factors for nuclear RNA localization. It is noteworthy that these screenings reveal both positive and negative regulators for long non-coding RNA (lncRNA), MALAT1 nuclear speckle localization, suggesting that lncRNA is located in the dynamic regulation of subcellular compartments.

[0179] These embodiments develop an imaging-based mixed library CRISPR screening approach that allows the phenotype and genotype of individual cells to be read out by high-resolution, high-content imaging. This approach promises to greatly expand the phenotypic space accessible to mixed genetic screening by allowing the detection of complex cellular phenotypes, such as cell morphology, subcellular organization of different molecular species, and their dynamics. This approach was applied to screen for genetic factors involved in nuclear RNA localization, identifying both positive and negative regulators that control lncRNA localization to nuclear spots.

[0180] These examples illustrate all imaging-based mixed library CRISPR screening methods in mammalian cells. This approach allows high-content phenotypic imaging of multiple molecular targets in a single cell and more accurate identification of the genotype of each cell, the latter by associating each sgRNA with a unique barcode and reading the barcode using multiple error robust fluorescence in situ hybridization (MERFISH) to achieve. In order to illustrate the capabilities of this method, genetic screening of factors that regulate RNA localization in nuclear compartments was performed. Various nuclear RNAs such as small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), and long noncoding RNAs (lncRNAs) are associated with nuclear compartments such as nucleoli and nuclear spots formed by liquid-liquid phase separation. Understanding the spatial regulation of these RNAs is important for understanding how they organize diverse nuclear activities and functions, including transcriptional regulation, transcriptional processing, and genomic stability. The effects of 162 sgRNAs (targeting 54 genes) were screened for the localization of 6 RNA targets, including: lncRNA MALAT1, U2 snRNA, and noncoding RNA 7SK, all of which are known to localize to nuclear speckles; nascent preribosomal RNA and noncoding RNA MRP, both of which are known to localize to the nucleolus; and polyadenylated RNA. These results reveal some regulators for nuclear RNA localization. Specifically, two positive regulators that are critical for nuclear speckle localization of MALAT1 and negative regulators that reduce nuclear speckle localization of MALAT1 were identified, suggesting that lncRNA localization is dynamically regulated.

[0181] Example 2

[0182] This example illustrates high-throughput, high-precision barcode imaging in mammalian cells. In situ imaging-based mixed library screening has been performed in bacteria in recent years, where the genotype of a single cell is identified by multiplexed FISH imaging of barcodes associated with gene variants. Because bacterial cells are small in size, the diffusion signal from the barcode RNA in a single cell is strong enough to be easily measured. However, mammalian cells are about a thousand times larger in volume than bacteria, making it difficult to achieve a high enough concentration of barcode RNA to allow reliable measurement. Therefore, new barcode expression and detection schemes are needed for mammalian cells to increase barcode signals and reduce background.

[0183] To achieve this goal, two independent promoters were used to express the sgRNA and reporter gene in the same vector and a 12-bit ternary barcode was incorporated into the 3' untranslated region (3'UTR) of the reporter gene ( Figure 1AEach digit of the ternary barcode (hereafter referred to as a trit) is made from one of three different read sequences (30 nucleotides (nt) long) specific to that digit, corresponding to three possible trit values ​​0, 1, and 2. For example, 12 trits have a total encoding of 3 12 =531,441 barcodes. Because there are 36 different triple sequences in total (3 different sequences in each of the 12 triples), the barcodes are read using sequential rounds of hybridization to form an image with 36 pseudo-color channels (18 rounds of hybridization, 2-color imaging per round, one pseudo-color channel per triple sequence), providing highly multiplexed detection. To increase the signal from the barcodes, a branched DNA amplification scheme was used to amplify the signal of each triple sequence ( Figure 1A To reduce interference from the background, the mRNA sequence of the reporter gene was co-stained, and both the reporter gene sequence and the barcode sequence were detected using single-molecule FISH (smFISH), so that only the barcode signal co-localized with the reporter gene signal was considered ( Figure 1A ). For each specific triple, a numerical value (0, 1, or 2) is assigned to the triple based on the pseudo-color channel that exhibits the highest proportion of reporter mRNA smFISH signals colocalized with the triple signal. This detection scheme reduces the background signal generated by nonspecific binding of the barcode FISH probe, which is important for the decoding accuracy shown in the following sections.

[0184] To test this barcoding scheme, a library of vectors was cloned, each containing in a mixed fashion a common reporter gene (luciferase-mCherry) and a unique barcode ( Figure 1B ; see below and Figure 7 ). Although the total number of possible barcodes exceeds 500,000, the library is limited to only ~2000 vectors (for error detection purposes, as described below), and the barcodes in the library are determined by sequencing. The library is delivered to the genome of U-2OS cells using lentivirus at a low multiplicity of infection (MOI) so that most infected cells receive only one barcode. The barcode signal of a single cell is measured using a multiplex detection scheme as described above.

[0185] After each round of hybridization, bright barcode signals co-localized with the smFISH signals of the reporter gene (luciferase-mCherry) mRNA were observed ( Figure 1C For each three-bit assay, the three three-bit values ​​were probed separately (in different pseudo-color channels as described earlier) and three distinct cell populations were observed, representing cells expressing the barcodes for the three different three-bit values ​​( Figure 1D ; see below and Figure 8 ). The k-means clustering algorithm was used to separate the three cell populations, with each population assigned a three-bit value based on which of the three pseudo-color channels assigned to that three-bit showed the highest proportion of reporter mRNA spots co-localized with the three-bit signal. Detection of 12 three-bits using 36 pseudo-color channels allowed the assignment of a barcode to each cell. The barcodes decoded for the majority of cells (~57%) matched the ~2000 barcodes in the library determined by sequencing ( Figure 1E ), discarding cells with mismatched barcodes. To assess the improvement in barcode detection accuracy using this reporter gene colocalization approach, the barcode for each cell was also assigned based on the number of FISH spots detected for individual barcode signals (without considering colocalization with reporter gene signals). It was found that in this case no decoded barcode matched an actual barcode in the library ( Figure 1F ), which is presumably due to background signals introduced by nonspecific FISH labeling, indicating that the use of reporter gene co-localization greatly improves decoding accuracy.

[0186] Using a bottleneck strategy (i.e., limiting the total number of vectors in the library to ~2000, which is only <0.4% of the possible total number of 12-bit ternary barcodes) allows for error detection, since a read error at any bit is most likely to generate an invalid barcode that is not present in the library. Quantitatively, since only 0.4% of all possible barcodes are present in the library, the probability that any incorrectly detected barcode matches a barcode in the library is only 0.4%. Therefore, of the 57% of barcodes that are exact matches, only 0.3% are likely to be from barcode misidentification (see detailed calculations below).

[0187] FIG1 shows imaging-based barcode detection for genotyping in mammalian cells. Figure 1A The purpose is to show a strategy for high-precision imaging-based barcode detection for genotyping. sgRNA and a reporter gene with an imaging-based barcode are co-delivered to the genome of the host cell. The reporter gene portion of the mRNA is detected by single-molecule FISH (smFISH), and the barcode is detected by MERFISH, using successive rounds of hybridization to detect each bit (three bits) of the ternary barcode. The barcode signal is amplified using a 4X4 branched DNA amplification scheme. Figure 1B Shown is the construct design of a reporter gene-barcode library used to probe the accuracy of barcode identification. Figure 1C, the upper figure shows an example image showing the reporter mRNA smFISH signal and the signal for each of the three three-digit values ​​(0, 1, and 2) for a single three-digit barcode. The lower figure shows an enlarged view of the white box area of ​​the upper figure, where the reporter gene signal is shown on the left and the overlap between the reporter gene signal and the barcode three-digit signal is shown on the right. For this cell, the three-digit value 1 has a high colocalization ratio, while the three-digit values ​​0 and 2 do not have a high colocalization ratio. The scale bar is 10 microns. Figure 1D Shown the colocalization ratio of three three-dimensional numerical values ​​of three measurements of all cells.Each spot in the mapping corresponds to a single cell.Colocalization is defined as the number of reporter gene smFISH spots colocalized with three signal spots divided by the sum of reporter gene smFISH spots in the cell.Based on its colocalization, use the k-means clustering algorithm to divide cells into three clusters (shown with different shades).Each cluster corresponds to the cell with specific three-dimensional numerical value. Figure 1E Shown is a histogram of the number of cells with different numbers of mismatched triplets in the decoded barcodes compared to valid barcodes in the library. Barcodes were decoded as described above using colocalization of reporter and triplet signals. Figure 1F and Figure 1E Same as, but employing barcodes that are decoded by using only the number of measured three-way signal spots, without taking into account the co-localization of the reporter gene signal and the three-way signal.

[0188] Figure 7 The library cloning strategy used to evaluate the accuracy of imaging-based barcode detection is shown. In brief, barcodes and UMIs (unique molecular identifiers) are first assembled from single DNA oligonucleotides by two-step overlapping PCR (see below). The shading of different oligonucleotides represents different three-bit sequences. This barcode-UMI library is then inserted into the digested lentiviral plasmid backbone to form a barcode-UMI lentiviral vector library. The reporter gene cassette is further inserted into the barcode-UMI lentiviral vector library to form the final reporter gene-barcode library.

[0189] Figure 8 Shown are all 12 three-dimensional colocalization ratio analysis.To all 12 three-dimensional display to all cells measured single three-dimensional three numerical value colocalization ratio.Based on its colocalization ratio using k-means clustering algorithm cells are divided into three clusters (shown with different shades).Each cluster corresponds to the cell with a three-dimensional numerical value.

[0190] Example 3

[0191] To further verify the low false identification rate, in this example, two reporter gene-barcode libraries were designed, each expressing the reporter gene luciferase-mCherry ( Figure 2A ), and the two libraries were cloned separately. Each library was reduced to contain <0.2% of all possible barcodes, making it very unlikely that the same barcode would appear in both libraries, and the identity of the barcode associated with each epitope-tagged reporter gene was determined by sequencing. The two libraries were introduced into U-2OS cells separately, and then the cells of the two libraries were mixed together in approximately equal numbers. The phenotype of each cell, i.e., the expression of HA or Myc tags, was imaged using immunofluorescence ( Figure 2B ), using sequential rounds of hybridization as described above to image the barcode associated with each cell. The rationale is that determining the phenotype of each cell will allow the barcode identity of that cell to be deduced from the sequencing results, which will then be compared to the barcodes determined by imaging to allow the identification of a small fraction of misidentified barcodes.

[0192] Judging by barcode-phenotype mismatch, only ∼1% of cells had incorrectly labeled barcodes ( Figure 2C and 2D ). Even this small error is likely due to errors in cell segmentation, which in turn leads to errors in phenotypic determination, further supporting the very low barcode misidentification rate in these experiments.

[0193] FIG2 illustrates an evaluation of the misidentification rate using barcodes with known phenotype-barcode correspondences. Figure 2A The constructs used to evaluate the accuracy of barcode detection are shown. The reporter gene luciferase-mCherry is tagged with HA or Myc tags to define two phenotypes, and a nuclear localization signal to concentrate HA and Myc signals in the nucleus to facilitate detection. The barcode is placed at the 3'UTR of the reporter gene, and the correspondence between the barcode and the HA or Myc tag is determined by sequencing. The reporter gene is driven by the CMV promoter. Figure 2B The images shown show HA and Myc immunostaining signals in two different channels. Cell nuclei with strong HA signals have weak Myc signals and vice versa. Cell borders are brightened. Nuclear borders of HA-expressing cells and Myc-expressing cells are also marked separately. Scale bar is 50 μm. Figure 2CIt is a scatter plot of the HA and Myc immunostaining intensity of a single cell. It shows cells that are assigned to HA or Myc libraries according to the barcode based on imaging. Cells that are classified as HA or Myc immunostaining positive are shown by triangles or circles respectively (see below). Only 10 of the 1105 cells with the barcode corresponding to HA were observed to be Myc immunostaining positive, and only 9 of the 1034 cells with the barcode corresponding to Myc were observed to be HA immunostaining positive, indicating that the barcode misidentification rate is low. The HA and Myc immunostaining signals of a small number of cells (197 in 2336 cells) are all below the threshold or their HA and Myc immunostaining signals are all above the threshold, so they are not clearly identified as HA positive or Myc positive (see below). These cells are excluded from the analysis. Figure 2D Shown are histograms of the ratio of HA intensity to Myc intensity for individual cells decoded by barcode imaging to contain either an HA-tagged reporter or a Myc-tagged reporter.

[0194] Example 4

[0195] This example illustrates a lentiviral delivery system with reduced recombination effects for accurate sgRNA labeling. Another challenge for sgRNA labeling by hybrid barcode imaging comes from the viral system used to deliver the sgRNA-reporter-barcode vector into mammalian cells. Lentivirus is a preferred delivery system for mammalian cells because it allows stable genomic integration of the vector and introduction of one sgRNA per cell by transfection at a low MOI (although other delivery systems can also be used in other cases), however, lentivirus has two single-stranded RNA genomes and is prone to recombination, which can lead to mispairing of sgRNA and barcode during viral transduction. The recombination rate of lentivirus is ~1 event per kilobase (kb). Because in these experiments, the sgRNA and reporter-barcode combination are expressed under the control of two independent promoters, respectively, the barcode and sgRNA sequences are separated by a large genomic distance (>1kb) in these examples, so the probability of recombination-induced barcode-sgRNA mispairing can be huge.

[0196] This example illustrates a strategy modified from the CROP-seq approach to overcome this recombination problem. Specifically, the reporter gene (puro-T2A-mCherry) was placed under the control of a strong Pol II promoter (EF1α, EF1-α), the sgRNA was placed under the control of an independent promoter (hU6), and the barcode was placed downstream of the polypurine band (PPT) in the lentiviral genome ( Figure 3A). In this way, the protospacer sequence (~20 nt sequence for specific gene targeting) and barcode sequence of the sgRNA can be separated by a minimal genomic distance (~100 bases). Although the expression of the sgRNA downstream of the reporter gene can be impaired by interference from the strong EF1α (EF1-alpha) promoter expressing the reporter gene, the sgRNA expression cassette is copied to the 5'LTR of the proviral genome during genomic integration, forming an additional functional unit to express the sgRNA without interference from the EF1α (EF1-alpha) promoter ( Figure 3A ). Transcription of the reporter gene terminates only at the 3' end of the 3'LTR, so the barcode should be expressed in the reporter gene mRNA 3'UTR for imaging-based barcode labeling ( Figure 3A ).

[0197] To evaluate whether this construct design can support functional lentiviral infection and sgRNA expression, a library containing both sgRNAs targeting genes essential for cell survival and non-targeting control sgRNAs was constructed. Efficient sgRNA expression can lead to cell depletion of sgRNAs targeting essential genes. 159 sgRNAs targeting 53 essential ribosomal proteins (3 sgRNAs per gene) and 51 non-targeting sgRNAs as controls were selected (dataset S1), and a lentiviral library containing these 210 sgRNAs, as well as a reporter gene (puro-T2A-mCherry) and a barcode was generated by mixed cloning ( Figure 3A ; see below and Figure S3). This lentiviral library was then used to infect U-2OS cells stably expressing Cas9-BFP. On the second day after lentiviral infection, cells infected with the library and expressing high levels of Cas9 were selected based on mCherry and BFP fluorescence, respectively, and these cells were kept for experiments at different time points after infection. The abundance of cells expressing various sgRNAs was determined by sequencing genomic DNA.

[0198] As expected, cells containing sgRNAs targeting essential genes were significantly depleted compared to cells containing non-targeting sgRNAs, and the extent of depletion depended on the length of time after lentiviral infection ( Figure 3B ), indicating that this viral system can support the expression of functional sgRNAs. In addition, the abundance of cells containing different sgRNAs was measured by imaging-based barcoding as described above. The abundance of cells containing individual sgRNAs measured by imaging-based barcoding correlated well with the abundance of cells measured by direct sgRNA protospacer sequencing ( Figure 3C ), further supporting accurate barcode detection.

[0199] Next, this experiment is used to evaluate the recombination rate of the construct. If recombination occurs, the barcode of the sgRNA assigned to the essential gene can be recombined with the non-targeting sgRNA, which should result in a higher cell abundance measured by barcode imaging than the cell abundance measured by protospacer sequencing. Similarly, the barcode assigned to the non-targeting sgRNA can be recombined with the sgRNA targeting the essential gene, resulting in a smaller cell abundance measured by barcode imaging. Therefore, for both cells containing sgRNA targeting the essential gene and cells containing non-targeting sgRNA, the fold change in relative cell abundance between day 2 and day 21 after lentiviral transduction was measured.

[0200] As expected, the relative abundance of cells containing sgRNAs targeting essential genes was greatly reduced on day 21 compared to day 2, while the relative abundance of cells containing non-targeting sgRNAs was greatly increased on day 21 ( Figure 3D Compared with the results obtained by sgRNA sequencing, the fold changes determined by barcode imaging were slightly smaller due to recombination ( Figure 3D This difference allowed quantification of the recombination-induced mispairing rate (see below), which was determined to be ∼8% between the sgRNA protospacer and the barcode ( Figure 3E ).

[0201] In addition, recombination-induced mispairing between the sgRNA protospacer sequence and a unique molecular identifier (UMI, a 20-nt sequence placed ∼500 bases downstream of the protospacer sequence) was measured. Figure 3A As expected, due to the larger genomic distance between the UMI and the protospacer (~500 nt), the recombination-induced mispairing rate in the region between the protospacer and the UMI was larger, ~16% ( Figure 3D and 3E). It is noteworthy that for random pairing of barcodes, the probability that these barcodes share the same sequence at any given three-position is about 1 / 3, because there are three possible sequences for any given three positions, and because the barcodes in the reduced library are a randomly selected subset of all possible barcodes. Therefore, it is estimated that the recombination in the barcode region should be roughly 1 / 3 of the recombination rate of fully homologous sequences of the same length. Based on the ~8% recombination rate measured for the ~100-nt genomic region between the barcode and the protospacer sequence (the common sequence of the sgRNA), the recombination rate in the ~400-nt barcode region is estimated to be approximately (400 / 100) x 8% / 3 = 10.7%, resulting in a recombination rate of ~8% + 10.7% = 18.7% for the genomic region between the protospacer sequence and the UMI, which is consistent with the measured value of ~16%. Furthermore, because the barcode library is reduced in size, recombination occurring within the barcode region is likely to generate new barcodes that match valid barcodes in the library, making barcode misidentification less likely.

[0202] In summary, the low error rate (<1%) in barcode imaging and the low mismatch rate (~8%) between sgRNA and barcode induced by recombination allow high accuracy in sgRNA identification by barcode imaging, which further allows for mixed library CRISPR screening based on full imaging. It is worth noting that although the remaining 8% mismatch rate between sgRNA and barcode can potentially generate false positives and false negatives in the screening, the error rate will be negligible because hundreds of cells carrying the same sgRNA are generally detected to determine which sgRNA has a statistically significant effect; moreover, three sgRNAs targeting each gene are detected, and only when two of the three sgRNAs show a statistically significant effect will the gene be considered a hit. Any remaining false positives can be easily identified by validation experiments.

[0203] Figure 3A Illustration of the design of a lentiviral delivery approach with low recombination-induced sgRNA-barcode mismatch rate. Figure 3A Lentiviral constructs used to deliver sgRNA and barcodes for sgRNA labeling are shown. The sgRNA cassette (hU6 promoter with sgRNA) and barcode array are placed downstream of the polypurine band (PPT). A strong Pol II promoter (EF1α, EF1-alpha) drives the expression of the reporter gene puro-T2A-mCherry. After genomic integration, the sgRNA cassette is copied to the 5'LTR for sgRNA expression, while the barcode and reporter gene are expressed at the 3'UTR for barcode imaging. UMI: Unique Molecular Identifier. Figure 3BProtospacer counts for each sgRNA at days 8, 21, and 28 after lentiviral transduction are shown plotted against protospacer counts measured at day 2 after transduction. Protospacer counts at days 8, 21, and 28 were normalized by a factor such that the average counts for non-targeting sgRNAs for these conditions were the same as the average counts for non-targeting sgRNAs at day 2. Protospacer counts were determined by sequencing. As expected, cells expressing sgRNAs targeting essential ribosomal genes were strongly depleted over time, so counts for sgRNAs targeting essential genes were greatly reduced compared to non-targeting control sgRNAs. Figure 3C Shown is the correlation between the number of cells expressing certain sgRNAs measured by imaging-based barcode detection and the sgRNA counts measured by protospacer sequencing at day 21 after lentiviral transduction. sgRNAs targeting essential genes are generally marked in red, and non-targeting control sgRNAs are generally marked in blue. Figure 3D is a violin plot showing the median fold change in relative sgRNA abundance between days 2 and 21 after lentiviral transduction as measured by protospacer sequencing, imaging-based barcode detection, and UMI sequencing. The relative abundance of a particular sgRNA is defined as the fraction of total sgRNA reads corresponding to this specific sgRNA (i.e., the number of protospacer (or UMI) reads determined by sequencing this particular sgRNA normalized to the total number of protospacer (or UMI) reads for all sgRNAs, or the number of cells expressing the barcode corresponding to this sgRNA determined by imaging normalized to the total number of cells). As expected, the relative abundance of sgRNAs targeting essential genes decreases over time, while the relative abundance of non-targeting sgRNAs increases. Due to recombination, the fold change determined by barcode imaging is slightly smaller than that determined by protospacer sequencing, and the fold change determined by UMI sequencing is slightly smaller than that determined by barcode imaging. Figure 3E Shown are the median mispairing rates between protospacer sequences and barcodes and between protospacer sequences and UMIs due to recombination determined at days 21 and 28 after lentiviral transduction. Error bars show 95% confidence intervals.

[0204] Example 5

[0205] This example illustrates a hybrid CRISPR screen for factors that regulate nuclear RNA localization. To demonstrate the power of this screening approach, potential regulators of RNA localization were screened in the nucleus ( Figure 4A). We selected 54 candidate genes involved in nuclear RNA regulation, including hnRNP family proteins, DExD / H box RNA helicases, and genes involved in RNA modification (Dataset S2). We designed a library of 167 sgRNAs, including three sgRNAs for each of the 54 genes and five non-targeting sgRNAs as controls, and generated a lentiviral library containing these sgRNAs as well as a reporter gene (puro-T2A-mCherry) and a barcode by mixed cloning (see below and Fig. 9 ). To demonstrate the ability of this method to evaluate complex phenotypes, the spatial distribution of five specific RNA species (lncRNA MALAT1, U2 snRNA, 7SK, MRP, and nascent pre-ribosomes, and polyadenylated RNA) was imaged using FISH. In addition, the nuclear speckle protein SON was also included in the phenotypic imaging using immunolabeling with oligonucleotide-conjugated antibodies. These RNA and protein targets were imaged using sequential rounds of hybridization, as well as barcode imaging, with 3-4 different color channels per round ( Figure 4A ) (See below for details of the imaging step).

[0206] As expected, the protein SON showed a clustered distribution marking nuclear speckles, and MRP and pre-ribosome signals marked subnucleolar compartments ( Figure 4B Based on these images, the boundaries of these structures were identified, and their number, the area they covered, and their average signal intensity (i.e., the total signal located within the identified cluster boundaries divided by the total area covered by these clusters) were determined in individual cells. Next, the enrichment of MALAT1, U2, 7SK, and poly-A-containing RNA in nuclear spots identified by SON staining was quantified (see below).

[0207] For each of these feature quantifications, the values ​​determined for cells carrying the targeting sgRNA were compared to the values ​​measured from cells carrying a non-targeting control sgRNA to determine the fold change. Four biological replicates of the experiment were performed, and a total of ∼30,000 cells were decoded, with hits determined based on the following criteria: at least two of the three sgRNAs targeting a gene exhibited a statistically significant fold change (Dataset S3).

[0208] As a positive control, a statistically significant decrease in cluster signal intensity, cluster area, and cluster number associated with SON staining was detected in cells expressing sgRNA targeting SON ( Figure 4C In addition, sgRNAs for several DExD / H-box RNA helicases (DDX10, DDX18, DDX21, DDX24, DDX52, and DDX56) resulted in statistically significant changes in various features of nascent preribosome staining ( Figure 4D), consistent with the known functions of these genes in ribosome biogenesis. Notably, the magnitude of the changes in these phenotypic traits was moderate ( Figure 4C and 4D ), potentially because not all cells expressing the sgRNA have their genomes edited. Thus, these quantifications allow identification of genetic perturbations with statistically significant effects, but the magnitude of the phenotypic change is less informative. It was also noted that perturbation of several genes in the hnRNP family resulted in significant changes in pre-ribosome and MRP signaling in the nucleolus (Dataset S3), potentially due to indirect effects.

[0209] Figure 4 shows an imaging-based hybrid CRISPR screen for regulators of nuclear RNA localization. Figure 4A The scheme of screening based on imaging is shown. Cells infected with lentivirus expressing sgRNA, barcode and reporter gene are fixed and imaged. Barcodes are imaged by MERFISH using 647-nm and 750-nm color channels in 18 rounds of hybridization (1-18 rounds). In order to increase the accuracy of barcode imaging, 561-nm color channels are used to image reporter mRNA in each round (1-18 rounds) to allow determination of the co-localization between barcode and reporter mRNA signals. In the first 7 rounds (1-7 rounds), 7 proteins and RNA targets for phenotypic measurement are imaged in 488-nm color channels. The mosaic on the left side includes 900 visual fields from a single screening. Figure 4B Phenotypic images of SON, MRP, pre-ribosomes, MALAT1, U2 snRNA, 7SK, and poly-A-containing RNA are shown. SON marks nuclear speckles, pre-ribosomes and MRP mark subnuclear structures. For SON, ribosomes, and MRP, the number of clusters, cluster area, and cluster intensity were quantified. For MALAT1, U2 snRNA, 7SK, and poly-A-containing RNA, their enrichment in nuclear speckles was quantified. Scale bar is 20 microns. Figure 4C Volcano plots showing the effect of each sgRNA on SON cluster intensity, cluster area, and cluster number. Figure 4D Volcano plot showing the effect of each sgRNA on pre-ribosome cluster intensity, cluster area, and cluster number. Figure 4C and 4DIn the , the fold change induced by each sgRNA was calculated as the mean of all cells containing this sgRNA divided by the mean of all cells containing non-targeting sgRNAs. The horizontal dashed line indicates the p-value (0.05) used to determine the hit of the screen. The data points of the indicated hits are shown in a shade matching the shade of the gene name shown in the legend (i.e., two of the three sgRNAs targeting this gene showed a statistically significant fold change (p<0.05)), and the data points of sgRNAs targeting other genes are shown in gray. The data points of non-targeting sgRNAs are shown in black.

[0210] Fig. 9 The cloning strategy of the lentiviral sgRNA-barcode delivery library is shown. In brief, barcodes and UMIs were first assembled from single DNA oligonucleotides by two-step overlapping PCR, and then assembled with protospacer sequences and sgRNA constant region sequences using overlapping PCR to form the sgRNA-barcode-UMI cassette library. The shading of different oligonucleotides represents different three-bit sequences. This library was then inserted into the digested reporter-containing lentiviral backbone with the hU6 promoter at a site downstream of the polypurine band (PTT).

[0211] Example 6

[0212] The present embodiment illustrates the new factor that participates in regulating the nuclear speckle localization of MALAT1. This screening reveals the gene (data set S3) that participates in regulating the nuclear speckle localization of different RNA species. Compared with 7SK, U2 snRNA and RNA containing polyadenylic acid, more genes that regulate the localization of MALAT1 are identified. This discussion focuses on MALAT1. Notably, two groups of genes ( Figure 5A ; Dataset S3), all but one gene (hnRNPH3) were validated by siRNA-mediated knockdown ( Figure 5B and 5C ). Due to the lack of effective antibodies for this protein, it has not been confirmed whether siRNA for hnRNPH3 is effective. Depletion of the first set of genes (DHX15, DDX42, hnRNPK, and hnRNPH1) resulted in a statistically significant reduction in the enrichment of MALAT1 in nuclear speckles ( Figures 5A-5C), indicating that these genes upregulate the nuclear speckle localization of MALAT1. DHX15 and DDX42 are involved in spliceosome recycling and assembly, respectively, which is consistent with the involvement of mRNA splicing factors in recruiting MALAT1 to nuclear speckles. The hnRNP family proteins hnRNPH1 and hnRNPK have not been previously reported to be involved in upregulating the nuclear speckle localization of MALAT1. These two genes were also found to affect the localization of other RNA species (including U2 snRNA, polyadenylated RNA, pre-ribosomal RNA and MRP) (Dataset S3), which may suggest a global effect of the perturbation of these two genes.

[0213] Surprisingly, three factors, hnRNPA1, hnRNPL, and PCBP1, were also identified that negatively regulated the nuclear speckle localization of MALAT1. Their depletion by sgRNA or siRNA induced a statistically significant increase in MALAT1 enrichment in nuclear speckles ( Figures 5A-5C The fold increase in MALAT1 enrichment induced by siRNA may be an underestimate due to incomplete knockdown. Combined knockdown of all three factors further increased MALAT1 enrichment in nuclear speckles (see below, Fig. 10A ), which, interestingly, also led to the enlargement of some of the nuclear speckles (see below, Fig. 10B and 10C The composition of each nuclear speckle (measured by the ratio of MALAT1 and SON levels in nuclear speckles) also became more diverse in the triple knockdown samples: some speckles had a reduced MALAT1 / SON ratio, while some had an increased ratio (see below, Fig. 10D These results suggest that enhanced nuclear speckle localization of MALAT1 after knockdown of the three negative regulators is associated with alterations in nuclear speckle morphology and composition. This suggests a role for MALAT1 in regulating nuclear speckle structure, consistent with the observation that MALAT1 knockdown can lead to reduced nuclear speckle size.

[0214] Figure 5 shows the genetic factors involved in regulating the nuclear speckle localization of MALAT1. Figure 5A The volcano plot of the effect of each sgRNA on the enrichment of MALAT1 nuclear spots is shown. The fold change is calculated as described in Figure 4. The horizontal dotted line represents the p value (0.05) used to determine the hit of the screening. The hits confirmed by siRNA knockdown are highlighted in the shadow matching the shadow of the gene name shown in the legend, and the data points of sgRNA targeting other genes are shown in gray. The data points of non-targeted sgRNA are shown in black. Figure 5BBox plots are shown showing the effect of siRNA knockdown of the seven hit genes on MALAT1 localization, next to data for control non-targeting siRNA. The middle line shows the median, the box shows the 25-75% quartile, and the whiskers show the maximum and minimum values. 100-300 cells were quantified for each condition. Student's t-test was performed for each condition compared to the control. ****, p<0.0001. Figure 5C Images of MALAT1 localization after siRNA knockdown of 7 hit genes are shown. Data from control non-targeting siRNA are also shown. MALAT1 staining is shown in the upper image and SON staining is shown in the lower image. Scale bar is 10 microns.

[0215] FIG. 10 shows that triple knockdown of hnRNPA1, hnRNPL and PCBP1 affects the morphology and composition of nuclear speckles. Fig. 10A Box plots are shown, showing the effects of control siRNA and single and triple knockdown (knockdown, KD) of HNRNPA1, HNRNPL and PCBP1 on MALAT1 localization. Box plot elements are as described in Figure 5. 100-300 cells were quantified for each condition. Student's t test was performed between each single KD and non-targeted control and between triple KD and single KD of hnRNPA1, hnRNPL or PCBP1. ****, p<0.0001. Fig. 10B Shown are example images of cells showing enlargement of some MALAT1-positive nuclear puncta (highlighted by arrows) after triple KD of hnRNPA1, hnRNPL and PCBP1 compared to cells transfected with control non-targeting siRNA. Scale bar is 10 microns. Fig. 10C The distribution of nuclear speckle sizes is shown, showing that triple KD of hnRNPA1, hnRNPL and PCBP1 increased nuclear speckle sizes. The difference between the two distributions was tested using a two-sample Kolmogorov-Smirnov test. Fig. 10D The log folds per nuclear speckle are shown for control siRNA and hnRNPA1, hnRNPL, and PCBP1 triple KD samples. 2 Distribution of (MALAT1 / SON intensity ratio). -300 cells and -7000 spots were measured for control and triple KD conditions, respectively.

[0216] Example 7

[0217] It has been previously suggested that nuclear speckle localization of MALAT1 may be impaired following transcriptional inhibition. However, the genetic factors involved in this process are largely unclear. Therefore, it was tested whether these negative regulators have a role in this process. To this end, in this example, the drug 5,6-dichloro-1-β(beta)-D-ribofuranosylbenzimidazole (DRB) was added to inhibit transcription, and a substantial reduction in MALAT1 enrichment in nuclear speckles was observed. Single knockdown of hnRNPA1, hnRNPL, and PCBP1 was largely unable to rescue DRB-induced dissociation of MALAT1 from nuclear speckles ( Fig. 6A and 11 On the other hand, double knockdown of two of the three factors or triple knockdown of all three factors largely rescued this DRB-induced dissociation effect ( Fig. 6A , 6B and 11, see also below), suggesting that these hnRNP family proteins are required for the dissociation of MALAT1 from nuclear speckles induced by transcriptional inhibition, and that these factors may play multiple roles in this process. These results provide a potential mechanism for the dissociation of MALAT1 from nuclear speckles by transcriptional inhibition. During transcriptional inhibition, RNA-binding proteins such as hnRNPA1 and hnRNPL are freed from nascent mRNA transcripts to allow them to bind to other RNA species. It is therefore possible that free hnRNPA1 and hnRNPL can bind to MALAT1, compete for factors that recruit MALAT1 to nuclear speckles, and thus prevent nuclear speckle localization of MALAT1 during transcriptional inhibition.

[0218] FIG. 6 shows that hnRNPA1, hnRNPL, and PCBP1 are required for transcriptional repression-induced dissociation of MALAT1 from nuclear speckles. Fig. 6A Shown in the presence or absence of transcription inhibitor DRB treatment (50 micromolar, 1h) to the quantification of MALAT1 nuclear speckle enrichment of cells transfected by different siRNA combinations. 100-300 cells were quantitatively analyzed for each condition. The MALAT1 induced by transcription inhibition can not be rescued from the dissociation of nuclear speckles by single knockdown of hnRNPA1, hnRNPL and PCBP1, but can be rescued by double knockdown and triple knockdown of these factors. Figure 6B Images are shown showing that in cells transfected with control siRNA, MALAT1 dissociates from nuclear speckles upon transcriptional inhibition, whereas in cells co-transfected with siRNA targeting hnRNPA1, hnRNPL, and PCBP1, transcriptional inhibition fails to dissociate MALAT1 from nuclear speckles. Scale bar 10 μm.

[0219] Fig.11The fold change of MALAT1 nuclear speckle enrichment after transcription inhibition under different knockdown conditions is shown. The bar graph shows the fold change of MALAT1 nuclear speckle enrichment after transcription inhibition under various knockdown conditions. This fold change is defined as the enrichment of MALAT1 nuclear speckles after transcription inhibition after the same siRNA treatment divided by the enrichment before transcription inhibition. Control siRNA, hnRNPA1, hnRNPL and PCBP1 single knockdown and hnRNPA1, hnRNPL and PCBP1 double knockdown and triple knockdown conditions are shown. Error bars: SD. Student's t test was performed for each condition relative to the control. **, p<0.01, ****, p<0.0001, ns, not significant. n=3, each experiment contained 30-100 cells.

[0220] Example 8

[0221] These embodiments develop an imaging-based mixed library CRISPR screening method that allows the establishment of genotype-phenotype correspondence for individual cells and allows high-throughput screening of mammalian cells based on complex phenotypes that could not be used in previous mixed library screening. The sgRNA identification detected by MERFISH-based barcodes allows this imaging-based screening, and the barcode misidentification rate is as low as ~1%. A lentiviral delivery scheme was designed in which the mismatching rate of recombinant-induced sgRNA and barcodes was reduced (mismatching rate <10%). In summary, these provide highly accurate sgRNA identification by barcode imaging. These methods greatly expand the phenotypic space available for mixed library screening. Compared with imaging-based screening using an array format (wherein a single gene perturbation is measured separately in a single well), the main advantage of performing mixed screening is that the reagents for gene perturbation, i.e., DNA plasmids and lentiviruses, can be prepared in a mixed manner using standard molecular biology operating procedures, requiring less labor and cost, which is particularly beneficial for large-scale custom designed libraries. Reagent preparation for array screening generally requires expensive multi-well robotic processing systems and more complex operating procedures. Another advantage of the hybrid approach is that the changes in experimental conditions for different perturbations can be minimized because all genetic perturbations are measured in the same experiment. This is particularly desirable when cells should be treated with concentration- or time-sensitive conditions. Moreover, the hybrid format can also simplify multiple phenotypic measurements, which require consecutive rounds of staining and signal removal by buffer exchange. On the other hand, array screening may be preferred when reagents for generating individual genetic perturbations are not particularly needed and when phenotypic measurements are not very sensitive to changes in sample processing conditions, because the MERFISH barcode readout process greatly increases the complexity of the imaging operating procedure.

[0222] Current 12-bit ternary barcode libraries contain over 500,000 barcodes. Even with a strict 1% bottleneck strategy for error-robust barcode detection, over 5,000 different sgRNAs can be included in each library, a capacity that can be easily increased by adding more bits to the barcodes. The current limitation on the number of sgRNAs that can be screened is the time required to image a large number of cells. This imaging system utilizes a high magnification (60X) objective to read out the FISH signal on each single mRNA molecule for barcode detection, limiting the number of cells that can be imaged in each field of view. However, imaging speed can be greatly improved by: 1) using greater magnification for barcode signals, allowing each field of view to be captured at a faster frame rate and / or by using lower magnification objectives, allowing more cells to be imaged in each field of view; 2) using multiple cameras for detection, allowing simultaneous detection of fluorescent signals in different color channels. With these improvements, a more than 10-fold increase in the number of cells and genotypes that can be screened per experiment can be achieved.

[0223] To demonstrate the efficacy of this approach for screening complex phenotypes in mammalian cells, the subcellular localization of seven different molecular species, including six RNAs and one protein, was imaged. These screening experiments revealed previously unknown regulators of nuclear RNA localization. Interestingly, positive and negative regulators of nuclear speckle localization of the lncRNA MALAT1 were identified. Positive regulators included DExD / H box RNA helicases, DHX15 and DDX42, and hnRNP family genes, hnRNPH1 and hnRNPK; while negative regulators included hnRNPA1, hnRNPL, and PCBP1. RNAs can be localized to cellular compartments formed by phase separation by at least two mechanisms: 1) RNAs can act as scaffolds that can nucleate phase separation, such as mRNAs in P bodies and stress granules and pre-ribosomal RNAs in the nucleolus; 2) RNAs can be recruited to phase separation bodies as clients, which have been shown to be responsible for the localization of MALAT1 in nuclear speckles. It is possible that the negative regulators found in this screen can compete with factors that recruit MALAT1 to nuclear speckles, thereby preventing nuclear speckle localization of MALAT1. The role of these negative regulators in the dissociation of MALAT1 from nuclear speckles induced by transcriptional inhibition was also identified. These results suggest that lncRNA localization can be dynamically regulated by protein factors.

[0224] These results demonstrate the ability of this imaging-based screening method to reveal molecular factors involved in cellular processes that can only be assessed by high-resolution imaging. This screening method can be widely used to investigate genetic factors that control or regulate a large number of phenotypes, including morphological features, the dynamics of molecular organization and cellular structure, and cell-cell interactions. This screening approach can also be combined with highly multiplexed DNA, RNA and protein imaging approaches, including genome-scale imaging approaches, to dissect the factors involved in gene regulation and other genome functions in a high-throughput manner.

[0225] Example 9

[0226] This example illustrates various materials and methods used in these examples.

[0227] The cloning of the reporter gene-barcode library and the sgRNA-reporter gene-barcode library was performed in a mixed manner using oligonucleotides ordered from IDT (datasets S1, S2, and S4). These libraries were cloned into the lentiviral vector pFUGW as described below. The identity of the barcodes present in the library and the barcode-sgRNA correspondence were established using high-throughput sequencing. Lenti-XTM Packaging Single Shots (VSV-G) (Takara, 631276) was used to prepare lentivirus in LentiX cells (Takara, 632180). U-2OS cells were infected with the lentiviral library at a low multiplicity of infection (MOI) so that only 10-20% of the cells were infected. Infected cells were sorted based on mCherry expression and Cas9-BFP expression. The sorted cells were fixed, permeabilized, and stained for imaging according to the detailed method discussed below.

[0228] A custom microscope built around a Nikon Ti-U microscope body with a Nikon CFIPlan Apo Lambda 60x oil immersion objective with 1.4NA was used for imaging. For sequential rounds of hybridization and imaging, a peristaltic pump (Gilson, MINIPULS 3) pumped liquid (TCEP buffer for stain lysis, hybridization buffer containing readout probes, or hybridization buffer for sample washing) into Bioptech's FCS2 flow chamber with a sample coverslip, using three valves (Hamilton, MVP, and HVXM 8-5) to select the input fluid (see below for details). Barcode decoding and phenotypic quantification based on acquired images are also described in detail below.

[0229] Cell culture. U-2OS cells were cultured at 37 ° C in EMEM medium (ATCC, HTB-96) supplemented with 10% FBS (Sigma, F4135-1L) and 1% Pen / Strep (Invitrogen, 15140122) antibiotics. U-2OS cells stably expressing Cas9-BFP were generated by lentiviral transduction, followed by FACS sorting using the BFP signal. To generate a lentiviral vector for Cas9-BFP, the Cas9-BFP sequence was PCR amplified from pLentiCas9-BFP (Addgene #78545) and cloned into the pFUGW backbone with the SVVF promoter. Two nuclear localization signal sequences were added to enhance the nuclear localization of Cas9.

[0230] Vector library cloning. Each 12-bit barcode consists of 12 30-nt sequences, each 30-nt sequence represents a three-bit, and the nucleotide 'A' separates adjacent three-bits. Oligonucleotides encoding each pair of 30-nt sequences adjacent in a forward and reverse alternating manner were ordered from IDT (i.e., trit1+trit2, trit2+trit3 reverse complement, trit3+trit4, trit4+trit5 reverse complement, ..., trit9+trit10, trit10+trit11 reverse complement, trit11+trit12, see data set S4). Each three-bit has three different values, identified by three distinct 30-nt sequences, so each pair of adjacent 30-nt sequences has 9 possible different sequences, and a total of 11x 9=99 oligonucleotides are required to cover all possible adjacent 30-nt sequence pairs. At both ends of the barcode (represented by oligonucleotides 1-9 and 91-99), two constant primer binding sequences are added for PCR amplification purposes. The sequences of these 99 oligonucleotides are recorded in Dataset S4. The entire barcode library was assembled by two-step overlapping PCR. First, the 12 bits were divided into 3 segments, each of which was generated by the following reaction: Segment 1. Oligonucleotides 1-36 as templates, oligonucleotides 1-9 as forward primers, and oligonucleotides 28-36 as reverse primers; Segment 2. Oligonucleotides 37-72 as templates, oligonucleotides 37-45 as forward primers, and oligonucleotides 64-72 as reverse primers; Segment 3. Oligonucleotides 73-99 as templates, oligonucleotides 73-81 as forward primers, and oligonucleotides 100 as reverse primers. The three PCR products were gel purified. Then, the three segments were mixed and overlapping PCR was performed using forward primer oligonucleotide 101 and reverse primer oligonucleotide 102. The reverse primer of this step contains a 20-base random sequence region that serves as a unique molecular identifier (UMI) for the sequencing step. The sequence of oligonucleotides 100-102 is also recorded in data set S4. All PCR reactions were performed using real-time qPCR equipment to monitor the reaction so that the reaction stopped in the logarithmic growth phase to reduce the library offset caused by PCR bias. The PCR products were assembled into modified pFUGW backbones by isothermal assembly. The assembled library was electroporated into Endura competent cells (Lucigen, 60242-2), which were then grown overnight under ampicillin selection to amplify the library. The amplified library was purified by mini prep. This library was named pFUGW_barcode_UMI_main chain library.

[0231] The pFUGW_barcode_UMI_main chain library is then used to generate a library that additionally comprises a reporter gene (luciferase-mCherry) for barcode imaging. First, a reporter gene cassette comprising a CMV promoter and a reporter gene open reading frame is generated in an intermediate vector. The open reading frame comprises luciferase-mCherry, 2X HA or 2X Myc tags at the N-terminus and a nuclear localization signal at the C-terminus. Oligonucleotides 103 and 104 (sequences are provided in data set S4) are used to PCR amplify the reporter gene cassette from the intermediate vector. The pFUGW_barcode_UMI_main chain library is then digested with BstXI and treated with alkaline phosphatase, and is assembled with the reporter gene cassette PCR product using isothermal assembly. The assembled library is electroporated into Endura competent cells, which are then grown overnight for amplification under ampicillin selection. The cells are then diluted to include the construct of the desired number in each library. These reduced libraries are then purified by mini prep. These libraries are named as reporter gene_barcode libraries.

[0232] Cloning of sgRNA-barcode library. The following strategy was used to clone the sgRNA-barcode library: first, a prototype spacer sequence-sgRNA constant region-barcode box library was generated by multi-step overlapping PCR; this library was then inserted into a lentiviral vector by isothermal assembly, in which the U6 promoter was placed downstream of the PPT sequence. In order to generate a prototype spacer sequence-sgRNA constant region-barcode box library, the barcode segment was first generated using a similar method as described in the "Cloning of barcode libraries for quantitative barcode decoding accuracy" section. The only difference is that the constant (primer) region at the 5' end was changed by replacing oligonucleotides 1-9 with oligonucleotides 105-113 because the barcode was placed next to the sgRNA. The sgRNA constant region was PCR amplified using oligonucleotides 114 and 115. The prototype spacer sequence library was ordered from IDT, where the constant region was used for PCR amplification on both sides of the prototype spacer sequence. In this work, two protospacer libraries were generated, one for essential ribosomal genes and non-targeting sgRNA controls to measure the recombination rate between sgRNA and barcode (dataset S1), and the other for targeting genes that potentially regulate RNA localization in the nucleus (dataset S2). The protospacer library was PCR amplified using oligonucleotides 116 and 117, and the PCR products were gel purified. The protospacer, sgRNA constant region, and barcode PCR products were then mixed and overlap PCR was performed using oligonucleotides 116 and 118 as primers. Reverse primer oligonucleotide 118 contained a 20-base random sequence region that served as a unique molecular identifier (UMI) for the sequencing step. The sequences of oligonucleotides 105-118 are also recorded in dataset S4. All PCR reactions were performed using a real-time qPCR device to monitor the reaction, so that the reaction was stopped in the logarithmic growth phase. The PCR products were assembled into a modified pFUGW backbone by isothermal assembly, in which the U6 promoter was placed downstream of the PPT sequence. The assembled library was electroporated into Endura competent cells, which were then grown overnight on ampicillin selection plates for amplification. A specific number of colonies (~3800 for essential ribosomal gene libraries, ~2500 for RNA positioning screening libraries) were scraped from the plate with LB buffer and cultured overnight in 200 mL LB buffer. The library was purified by mini prep. These libraries are named sgRNA_barcode libraries.

[0233] Sequencing library preparation and analysis. In order to determine the identity of the barcodes presented in the library and establish the correspondence between sgRNA and barcode, the library was analyzed using high-throughput sequencing. It was found that PCR amplification of the barcode region can lead to recombination of the barcode due to homology between barcodes. Therefore, a ligation-based approach was used to install sequencing adapters to the barcode library. In this approach, the region to be sequenced is digested from the library and then ligated to the adapter using T4 ligase.

[0234] To determine the barcodes in the reporter gene-barcode library, the library was digested with BstXI and BamHI at 37°C for 2-3 hours, and the resulting fragments were purified using the Zymo DNA purification kit (ZD4002). To generate adapters with sticky ends for ligation, oligonucleotides 119-124 (sequences provided in Dataset S4) were mixed at 0.5 micromolar each, subjected to 5 cycles of PCR reaction, and the products were purified using the Zymo DNA purification kit to produce double-stranded sequences with 5' and 3' adapters separated by BstXI and BamHI digestion sites. The purified product was digested with BstXI and BamHI at 37°C for 2-3 hours and then purified. The resulting mixture contained adapters with sticky ends for ligation and was mixed with the above-mentioned purified library fragment mixture. T4 ligase was added and the reaction was kept at room temperature for 2-4 hours. The reaction mixture was directly subjected to 2% agarose gel electrophoresis, and the band corresponding to the size of ~400 bp was cut and purified. The purified DNA samples were used for concentration measurement and high-throughput sequencing using the V2-MISeq kit (Illumina, MS-103-1003).

[0235] In order to determine the sgRNA-barcode correspondence of the sgRNA_barcode library, two sequencing libraries were generated because the length from the protospacer to the end of the barcode was greater than 500 bp, which exceeded the optimal length range for high-quality sequencing of the V2-MISeq kit. In one library, BstXI and BamHI were used to generate the junction site, which was located at the 5' of the protospacer and the 3' of the UMI, respectively. The sequencing of this library covered the protospacer region, a portion of the barcode region, and the UMI region, because sequencing from either end of this library may not reach the middle part of the barcode. In the second library, KpnI and BamHI were used to generate the junction site (the KpnI site was immediately after the sgRNA and before the barcode). The sequencing of this library covered the entire barcode region and the UMI region. The UMI sequence was used to identify the protospacer and barcode in the same construct from these two libraries. Oligonucleotides 125-130 and oligonucleotides 131-136 (sequences provided in Dataset S4) were used to generate aptamers for the first and second libraries, respectively. The procedure was the same as described for the generation of sequencing libraries for the reporter-barcode library.

[0236] UMI, protospacer sequence, and barcode sequence were extracted from sequencing reads. The reads were then grouped by common UMI and barcode to generate a codebook for sgRNA-barcode correspondence. Reads with incorrect protospacer sequences or barcodes assigned to multiple sgRNAs were removed from further analysis.

[0237] To determine the distribution of protospacer sequences and UMIs from cell populations at different time points after lentiviral transduction in the experiment using sequencing to determine recombination rates, sequencing libraries were prepared by PCR amplification from purified genomic DNA using oligonucleotides 137-140 and oligonucleotides 141-144 (sequences provided in Dataset S4) as forward and reverse primers, respectively.

[0238] Lenti-X cells (Takara, 632180) were used for lentiviral transduction. TM Packaging Single Shots (VSV-G) (Takara, 631276) was used to prepare lentivirus. The prepared virus was used with Lenti-X TMThe concentrate (Takara, 631231) was concentrated and stored at -80°C. For transfection, the amount of virus was controlled so that 10-30% of the cells were transduced to ensure that most infected cells were infected with only 1 viral particle. Viral transduction was performed using 10 μg / ml polybrene (Sigma, TR-1003-G). Compared with the construct without insertion after PPT, the viral titer of the construct with U6-sgRNA-barcode array placed after PPT did not show a significant reduction, indicating that the insertion did not impair lentiviral transduction.

[0239] siRNA knockdown. All siRNAs were purchased from Dharmacon, and siRNA knockdown was performed according to the experimental flow of Dharmacon. In brief, U-2OS cells were seeded on imaging coverslips in 12-well plates at 30,000 cells / well. For siRNA transfection, 1.5 microliters of 20 micromolar siRNA were added to 100 microliters of serum-free, antibiotic-free culture medium in a test tube, and 1 microliter of Dharmacon transfection reagent (Dharmacon, T-2001-01) was added to 100 microliters of serum-free culture medium in another test tube. The two test tubes were incubated for 5 minutes and then gently mixed and incubated for another 20 minutes at room temperature. 800 microliters of antibiotic-free serum-containing culture medium were mixed into 200 microliters of the above-mentioned siRNA and transfection reagent mixture to generate 1 mL of transfection culture medium. Cell culture medium was replaced with 1 mL of transfection culture medium. Cells were incubated at 37 ° C for 72 hours before phenotypic measurement.

[0240] Silanization of imaging coverslips. The imaging coverslips were first cleaned with 1M KOH and pure methanol, washed with 70% ethanol, and dried in an oven. For silanization, the coverslips were soaked in silanization buffer (500 mL distilled water, 1500 microliters of affinity silane (Bind-silane, Sigma, GE17-1330-01), pH adjusted to 3.5 with glacial acetic acid) at room temperature for 1 hour. The coverslips were then washed with water and stored dry. Before inoculating cells, silanized coverslips were coated with 1% poly-D-lysine (Sigma, P0899) in a 60 mm diameter cell culture dish for 30 min, followed by a 1 hour wash with water.

[0241] Imaging sample preparation. U-2OS cells were seeded on coverslips two days before fixation. For phenotypic imaging in experiments screening for factors involved in regulating nuclear RNA localization, U-2OS cells were fixed 6 days after lentiviral transduction. Samples were fixed with 4% paraformaldehyde (EMS, 15714) in PBS for 15 min and permeabilized in 0.5% Triton-X (Sigma, X100) for 30 min. Next, samples were incubated in blocking buffer (500 μl blocking buffer: 50 μl 10x PBS, 200 μl RNase-free BSA (ThermoFisher, AM2618), 50 μl 25 mg / ml yeast tRNA (ThermoFisher, 15401029), 5 μl mouse RNase inhibitor (NEB, M0314L), 1 μl 25% Triton-X and RNase-free water to 500 μl) for 1 hour and stained with 1:100 primary antibody, anti-SON (Abcam, ab121759) in blocking buffer for 1 hour at room temperature. Samples were washed three times with 1xPBS and incubated with 1:300 oligonucleotide-labeled secondary antibody for 1 hour. Oligonucleotide-labeled secondary antibodies can later be detected by readout probes using sequence complementarity to the oligonucleotide sequence on the antibody. Samples were washed three times with 1xPBS and post-fixed with 4% PFA for 30 minutes. The samples were then equilibrated in 30% formamide in 2x SSC for 5 minutes before FISH staining. The FISH hybridization buffer contained 30% formamide (ThermoFisher, AM9342), 60% stellaris RNA FISH hybridization buffer (Biosearch, SMF-HB1-10), 10% 25mg / mL yeast tRNA and 1:100 mouse RNase inhibitor. The samples were stained overnight at 37°C with 300nM FISH probes for reporter genes, 300nM FISH probes for RNA phenotype (i.e., 6 RNA species) imaging, and 100nM primary amplification probes for barcode imaging. The FISH probes for reporter genes each contain a 30-nt targeting sequence that can bind to the reporter gene mRNA and 3 20-nt readout sequences that allow binding to complementary fluorescently labeled readout probes. Each FISH probe for each RNA target used in phenotypic imaging contains a 30-nt targeting sequence that binds to the RNA target and one or two 20-nt readout sequences that allow binding to complementary fluorescently labeled readout probes. Each primary amplification probe used for barcode imaging contains a 30-nt targeting sequence that binds to one of the 30-nt three-bit sequences on the barcode, and four additional 30-nt identical sequences that allow binding to the secondary amplification probes ( Figure 1A). The samples were then washed twice in 30% formamide in 2x SSC and stained with 100 nM secondary amplification probes for barcode imaging in 10% hybridization buffer (10% formamide, 80% stellaris RNA FISH hybridization buffer, 10% 25 mg / mL yeast tRNA and 1:100 mouse RNase inhibitor) at 37°C for 1 hour. Each secondary amplification probe contains a 30-nt target sequence that can bind to the primary amplification probe, and 4 additional 20-nt identical readout sequences that allow binding to complementary fluorescently labeled readout probes. Therefore, this amplification protocol allows a maximum of 16-fold signal amplification. Samples labeled with FISH probes for phenotypic imaging and reporter gene mRNA imaging and primary and secondary amplification probes for barcode imaging were washed twice in 30% formamide in 2x SSC and then embedded in 4% polyacrylamide gels, followed by incubation with protein digestion buffer (for 50 mL digestion buffer: 5 mL 8 M guanidine-HCL (ThermoFisher, 24115), 2.5 mL 1 M Tris pH 8.0 (ThermoFisher, 15569025), 100 microliters 0.5 M EDTA (ThermoFisher, 15575020), 0.25 mL Triton-X and 1:100 proteinase K (ThermoFisher, AM2548)) at 37°C overnight to remove proteins and lipids from the samples. This step is referred to as the sample washing step below. Proteinase K cleavage leads to protein digestion (including digestion of mCherry protein), so mCherry fluorescence signal disappears after digestion, and does not interfere with FISH signal detection using 561nm channel. FISH probes for RNA, 7SK, MRP, U2 snRNA containing polyadenylic acid and oligonucleotides connected to the second antibody for SON staining are conjugated with acrydite, and during the sample cleaning step, they can be cross-linked with polyacrylamide gel and these probes and the RNA they bind are retained in the gel. FISH probes for MALAT1 and pre-ribosomes are not labeled by acrydite, because the size of both MALAT1 and pre-ribosomes is large, so they are retained in the gel during sample cleaning. Reporter gene mRNA is connected to gel by poly-T probes (the probe can be bound to the poly-A tail of reporter gene mRNA) labeled by acrydite, thereby allowing FISH probes for reporter genes and FISH probes for barcode imaging to remain in the gel during cleaning. The sample cleaning step significantly reduces the background signal caused by the non-specific binding of cell autofluorescence and FISH probes to proteins and lipids. Samples were then washed with 2x SSC and placed in 2x SSC for imaging. The sequences of the FISH probes used are listed in Dataset S5.

[0242] For experiments to measure barcode labeling errors using two known phenotypes (expression of HA or Myc-tagged reporter genes), U-2OS cells were fixed 6 days after transduction. The tags were stained with primary antibodies (anti-Myc (Abcam, ab9132), anti-HA (Abcam, ab9110)), followed by staining with Alexa 405-labeled anti-mouse secondary antibodies (Abcam, ab175658) and Alexa 488-labeled anti-rabbit secondary antibodies (Invitrogen, A21206). Before gel embedding, samples were incubated in 25mMMA-NHS (Sigma, 730300) in 2x SSC for 1 hour, so that the MA-NHS-labeled antibodies were attached to the gel during gel polymerization. After sample washing, the antibodies were digested into fragments and the dyes were attached to the gel through the cross-linked antibody fragments. The dyes Alexa 405 and Alexa 488 can withstand polymerization during gel embedding. The rest of the sample preparation, including immunostaining and barcode staining, was as described above.

[0243] Antibody labeling by oligonucleotides. Antibodies were labeled with oligonucleotides using the following strategy. The antibody was first mixed with DBCO-NHS, which conjugated DBCO to the antibody, and then the DBCO-labeled antibody was mixed with an azide-labeled oligonucleotide to conjugate the oligonucleotide to the antibody. Specifically, 100 μg of anti-rabbit antibody (ThermoFisher, 31210) was first buffer exchanged into 100 μl of PBS using a 50KD protein concentrator (Millipore, UFC510024). NaHCO 3 And DBCO-NHS ester (Kerafast, FCC310) were added to the antibody solution so that their final concentrations were 50mM and 100mM, respectively. The reaction was allowed to continue at room temperature for 1 hour to prepare DBCO-labeled antibodies, and excess DBCO was removed by buffer exchange with PBS using a 50KD protein concentrator. Then PBS buffer was added to the DBCO-labeled antibody to make the solution volume reach 100 microliters, and 25 microliters of azide-labeled oligonucleotides (100 micromoles, data set S3) were added. The reaction was continued at 4°C overnight. After the reaction was completed, excess oligonucleotides were removed by buffer exchange using PBS, and the final oligonucleotide-labeled antibodies were aliquoted and stored at -80°C.

[0244] Imaging settings and continuous imaging. Imaging settings are as described above. See, for example, U.S. Patent Application Publication No. 2017-0220733 or International Patent Application Publication No. WO 2018 / 089438, the entire contents of each of which are incorporated herein by reference. In brief, a peristaltic pump (Gilson, MINIPULS 3) pumps liquid into a Bioptech's FCS2 flow chamber with a sample cover glass, and three valves (Hamilton, MVP, and HVXM 8-5) are used to select input fluids. A custom microscope built around a Nikon Ti-U microscope body is used for imaging, and the Nikon Ti-U microscope body is equipped with a Nikon CFIPlan ApoLambda 60x oil immersion objective lens with 1.4NA. Solid-state single-mode lasers (405 nm laser, Obis 405 nm LX 200 mW, Coherent; 488 nm laser, Genesis MX488-1000, Coherent; 560 nm laser, 2RU-VFL-P-2000-560-B1R, MPB Communications; 647 nm laser, 2RU-VFL-P-1500-647-B1R, MPB Communication; and 750 nm laser, 2RU-VFL-P-500-750-B1R, MPB Communications) were used for illumination. Acousto-optic tunable filters (AOTFs) were used to control the intensity of the 488 nm, 560 nm, and 647 nm lasers; the 405 nm laser was modulated by a direct digital signal; and the 750 nm laser was switched by a mechanical shutter. A custom dichroic (Chroma, zy405 / 488 / 561 / 647 / 752RP-UF1) and emission filter (Chroma, ZET405 / 488 / 461 / 647-656 / 752m) were used to separate the excitation illumination from the fluorescence emission. The emission light was imaged onto a Hamamatsu digital CMOS camera. During acquisition, samples were transferred using an automated XY stage (Ludl, BioPrecision2) and kept in focus using a homemade autofocus system.

[0245] For the experiment that needs higher imaging flux, use 40x object lens (CFI60 Plan Fluor40x oil immersion objective lens) to obtain more cells per visual field (FOV).Use four camera systems to separately and simultaneously gather the signal from 750nm, 647nm, 561nm and 488nm fluorophore.In detail, four camera mounts (QuadCam LS1.0x, 89North) are installed on Nikon Ti-U microscope body, and 4 Hamamatsu digital CMOS cameras are installed on the mount.4 dichroic filters (T495lpxr, T562lpxr, T647lpxr, T760lpxr, Chroma Tech) and 4 single-band emission filters (ET 450 / 50m, ET 525 / 50m, ET 605 / 75m and ET 705 / 70m, Chroma Tech) of 4 separations of the signal from different fluorophores are installed on the camera mount. In order to align the signal from 4 cameras, multicolor beads (FP-0257-2, Spherotech) are imaged and the signal from 647nm, 561nm and 488nm color channels is aligned with the signal from 750nm color channel by the cp2tform function in MatLab.Cp2tform derives polynomial spatial transformation to x, y and z coordinates, and the transformation is applied to barcode and phenotypic signals.In order to image the nucleus on the four camera settings, 1:1000 647nm Nucred dye (R37106, ThermoFisher) is used instead of DAPI.

[0246] Before loading into the flow cell, the sample was stained with an Atto565-labeled 20-nt readout probe (dataset S5) having a sequence complementary to the readout sequence on the FISH probe for reporter mRNA imaging. The staining was performed in hybridization buffer (10% ethylene carbonate (Sigma, E26258) in 2x SSC) at a readout probe concentration of 3 nM. The readout probe for the reporter gene was introduced only once, but imaging was performed repeatedly during all hybridization rounds. Readout probes for 7 molecular targets (SON proteins and 6 RNA targets) for phenotypic imaging and readout probes for barcode imaging were introduced in consecutive rounds of hybridization. For phenotypic and barcode imaging, 3 nM of 20-nt readout probes complementary to the oligonucleotide sequence on the SON antibody (Abcam, ab121759), or to the readout probes on the FISH probes for 6 RNA targets, or to the readout sequence on the secondary amplification probes for barcode imaging (Bio-Synthesis Inc., Dataset S5) in hybridization buffer (10% ethylene carbonate in 2x SSC) were flowed into the flow cell and allowed to stand for 15 minutes, followed by washing with hybridization buffer. The dyes for these probes, Alexa 488, Cy5, or Alexa 750, were attached to the oligonucleotides via cleavable disulfide bonds (Biosynthesis, Dataset S5). Samples were imaged in anti-bleach buffer (for 50 mL anti-bleach buffer: 50 mg glucose oxidase (gluco-oxidase, Sigma, G2133), 50 mg (+ / -)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (Sigma, 238813), 300 microliters catalase (Sigma, C100-500MG), 10% w / v glucose (Sigma, G8270), 5 mL 500 micromolar Trolox quinone and 50 microliters murine RNase inhibitor). For each round of hybridization, fluorescent signals from four color channels (488 nm, 561 nm, 647 nm and 750 nm if phenotypic imaging was included in that round) or three color channels (561 nm, 647 nm and 750 nm if phenotypic imaging was not included in that round) were imaged. After each round, the dye on the readout probe was cleaved by 10% tris(2-carboxyethyl)phosphine (TCEP; Sigma, 646547-10X1ML) followed by the next round of hybridization of the readout probe.

[0247] For the quantitative colocalization ratio between reporter gene signal and barcode signal and for the reason of image registration, 561nm channel detection reporter gene mRNA signal is used in each round.By continuous rounds of hybridization, and in 1-18 rounds, 647nm and 750nm channel imaging is used using cleavable Cy5 and Alexa750 dye, barcode signal is measured, which allows all 36 numerical values ​​of 12-three barcodes to be imaged.By continuous rounds of hybridization, and in 1-7 rounds, 488nm channel imaging is used using cleavable Alexa 488 dye, the signal of SON and 6 RNA targets in phenotypic imaging is measured.For phenotypic imaging, images are collected with a slightly higher focal plane (2-3 micron), which is most suitable for the signal from the nuclear interior.

[0248] For experiments measuring barcoding errors using two known phenotypes (expression of HA- or Myc-tagged reporter genes), Myc and HA tags were stained with Alexa 405-dye and Alexa 488-dye labeled secondary antibodies and imaged in the 405 nm and 488 nm channels, respectively.

[0249] DAPI staining was imaged and used for cell segmentation and nuclear identification. For experiments measuring two known phenotypes (HA or Myc-tagged reporters), DAPI staining was imaged during the last round of imaging. For experiments screening for factors that regulate nuclear RNA localization, DAPI staining was imaged during the first round of imaging. Sequences of dye-labeled readout probes are listed in Dataset S5.

[0250] Transcriptional inhibition. For transcriptional inhibition, 50 micromolar DRB (Sigma, D1916-10MG) was mixed in EMEM and incubated with cells for 1 hour before fixation.

[0251] Barcode decoding analysis. In order to correct the inconsistency in illumination, each image of the specified color channel is divided by the average intensity image of all images of the illumination color. Multiple rounds of images are registered using the uncleaved signal of reporter gene mRNA. Using DAPI staining as seed and cell autofluorescence (for evaluating barcode decoding accuracy and slow virus recombination experiments) or RNA staining containing polyadenylic acid for cell boundary identification (for screening experiments of factors regulating nuclear RNA localization) cells are segmented by watershed algorithm. Spot finding algorithm is used to identify the single molecule signal of reporter gene mRNA and barcode in all hybridizations. For experiments using 4-camera imaging, a segmentation algorithm is used to identify spots. In detail, pixels with intensity greater than the brightness threshold are selected. The cluster of selected pixels is identified by the bwareaopen function in MatLab. Clusters within the boundary area range (2-30 pixels) are retained. The area range is determined by visual inspection of the original image. In order to capture spots with intensity variations, this process is repeated using multiple brightness thresholds, for example, from 0.6x max (pixel intensity in FOV) to 0.3x max (pixel intensity in FOV), decreasing by 0.05x max (pixel intensity in FOV). The brightness threshold of each three-dimensional signal is manually determined. For each repetition, a lower brightness threshold will identify two types of clusters: (i) fuzzy clusters that cannot be detected under the higher brightness threshold of the previous round and (ii) larger clusters that completely include one or more clusters identified in the previous round. For any cluster of type (i), it is retained only if its area is within the above-mentioned allowable area range. For any cluster of type (ii), if its area is within the allowable area range, it is retained; otherwise, it is removed and the smaller clusters identified in the previous round that overlap with this new cluster are retained instead. The centers of these clusters are identified by the regionprops function in MatLab.

[0252] Single molecule FISH spots are assigned to cells, and the colocalization ratio of each of the three values ​​of the three digits in the barcode is calculated as the number of reporter gene smFISH spots colocalized with the barcode smFISH signal divided by the total number of reporter gene smFISH spots in the cell. In order to determine the numerical value of each three digits of each cell, cells are clustered by k-means clustering based on the three colocalization ratios of the three digits, and the three digit numerical values ​​are assigned to each cluster based on which of the three average colocalization ratios is the highest in the cluster. The same process is repeated for all 12 three digits so that each cell is assigned a 12-three digit barcode. For each numerical value of the three digits, the average colocalization ratio of the cell population assigned to the value is measured to be 0.4; and the average colocalization ratio produced by the random colocalization of the probe with non-specific binding, which is evaluated from two cell populations that are not assigned the three digit numerical values, is measured to be 0.1.

[0253] To decode cells based on barcode signals alone (i.e., without considering colocalization between barcode signals and reporter gene signals), cells were clustered based on the number of barcode signal spots detected for the three three-dimensional values ​​within each cell. The cells were divided into three populations using a k-means clustering algorithm, with the three-dimensional value assigned to each population based on which of the three three-dimensional values ​​had the highest average number of spots. This same process was repeated for all 12 three-dimensional values.

[0254] To estimate the barcode misidentification rate, since there are only 0.4% of all possible barcodes in the library due to the bottleneck strategy, the probability of any incorrectly decoded barcode matching a barcode in the library is only 0.4%. Therefore, of the 57% of exact matching barcodes, only approximately p = 0.3% can come from barcode misidentification (solved from (p*57%) / (1-57%) = 0.4% / (1-0.4%)).

[0255] Quantification of Myc and HA signals. To quantify HA and Myc expression in the nucleus, the nuclear border of each cell was used as a mask to measure the intensity of the corresponding Myc or HA channel. In order to allow HA and Myc expression to be clearly assigned to individual cells, thresholds for HA and Myc expression were first determined, above which HA or Myc tag expression could be detected with certainty. To determine these thresholds, the k-means clustering algorithm was used to cluster cells into two groups based on their non-thresholded HA and Myc tag staining intensities. This grouping allows the cells to be approximately divided into cells expressing HA and cells expressing Myc. In order to assess the background staining level of the HA tag, the mean and standard deviation of the HA intensity of the cells in the Myc-expressing clusters were calculated, and the threshold of the HA signal was calculated as the mean plus three standard deviations. The threshold for Myc expression was determined similarly to the HA-expressing clusters. Cells whose HA and Myc intensities were both below their respective thresholds or both above their respective thresholds were eliminated (197 of 2336 cells). After removing these ambiguous cells, the remaining cells were clustered again using the k-means clustering algorithm to obtain the final grouping, as shown in Figure 2C as shown in .

[0256] Recombination rate calculation. The recombination rate ai for the i-th sgRNA (with barcode or UMI) is based on the following formula:

[0257] B i，day n =P i，day 2 *((1-a i )*S i +a i *C)

[0258]

[0259] where n is the number of days after transduction, which in these experiments was equal to 21 or 28. iday 2 is the normalized protospacer read count determined by sequencing the i-th sgRNA on day 2 post-transduction (normalized by the total protospacer read count measured on day 2 post-transduction). P i，day n is the normalized protospacer read count determined by sequencing the ith sgRNA on day n after transduction (normalized by the total protospacer read count measured on day n after transduction). B i，day n is the normalized cell number determined by barcode imaging or the normalized UMI count determined by sequencing (normalized by the total cell number or UMI count at day n after transduction). i is the survival rate of the ith sgRNA. C is the average survival rate of all sgRNAs in the library, calculated by considering the redundancy weights of different sgRNAs in the library, and is the average survival rate if recombination occurs.

[0260] Phenotypic measurements are quantitative. Nuclear boundaries are determined by DAPI signal. Cells that the core contacts with the edge of the imaging field of view are excluded from further analysis. In order to identify the clusters of MRP, pre-ribosomes and SON, the background intensity of the channel is subtracted, and clusters are identified using functions regionprops (MatLab) and bwareaopen (MatLab), which are similar to the spot finding algorithm for the experiment of utilizing 4-camera imaging. In detail, the pixel whose intensity is greater than the brightness threshold is selected. The cluster of the selected pixel is identified by the bwareaopen function. The cluster in the boundary area range (20-3000 pixels for SON, 100-5000 pixels for pre-ribosomes, and 100-6000 pixels for MRP) is retained. The area range is determined by visual inspection of the original image. In order to capture the relatively wide clusters of the variation of the staining level, multiple brightness thresholds are used to repeatedly perform this process (from 0.9x max (pixel intensity in the core) to 0.1x max (pixel intensity in the core), decreasing by 0.05x max (pixel intensity in the core)). The number of the cluster of final mark and the area of ​​each cluster are measured using regionprops function.For MRP, preribosome and SON, the number of clusters, the average area of ​​clusters and the cluster intensity (defined as the total signal in the cluster border divided by the total cluster area) of each cell calculation cluster.For quantitative nuclear spot enrichment MALAT1, 7SK, U2 and the RNA comprising poly-A, the cluster border from SON dyeing is used as a mask to measure the MALAT1, 7SK, U2 and the RNA signal comprising poly-A in the described SON cluster border.In these RNAs, the nuclear spot intensity of each is measured as the total signal of the RNA described in the described SON cluster border divided by the total area covered by the SON cluster.The signal intensity outside the spot is measured as the total signal of the RNA in the core but outside the nuclear spot divided by the total area of ​​the core not in the nuclear spot.Nuclear spot enrichment is determined as the ratio of the signal intensity outside the nuclear spot intensity and the spot.

[0261] In order to identify the hits from this screening, the four replicates of the experiment were merged. The above quantitative values ​​(i.e., cluster number, cluster area, cluster intensity, and nuclear spot enrichment) of each repeat were normalized by the mean value of all cells in each repeat before merging. The p value was calculated by testing the measured values ​​of cells carrying a targeting sgRNA relative to the measured values ​​of cells carrying all control non-targeting sgRNAs using the Student's t test. When at least two sgRNAs targeting a specific gene show a p value <0.05, the gene is listed as a hit. SgRNAs with less than 40 cells are excluded from the analysis.

[0262] Dataset S1

[0263] sgRNA library for evaluating lentiviral design with respect to reduced recombination effects. This dataset lists oligonucleotide sequences of 159 sgRNAs targeting essential ribosomal genes and 51 protospacer sequences of non-targeting sgRNAs.

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273] Dataset S2

[0274] sgRNA library for genetic screening of factors regulating RNA localization in the nucleus. This dataset lists the oligonucleotide sequences of 162 sgRNAs targeting selected candidate genes for regulating RNA localization in the nucleus and the protospacer sequences of 5 non-targeting sgRNAs.

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282] Dataset S3

[0283] Features quantified and gene hits identified in a screen for factors regulating nuclear RNA localization. This dataset lists gene hits for individual phenotypic features quantified in the screen.

[0284]

[0285]

[0286] Dataset S4

[0287] DNA oligonucleotide sequences used for library cloning and sequencing. This dataset lists the DNA oligonucleotide sequences used for library construction and next-generation sequencing (for determining barcode identity and barcode-sgRNA correspondence within the library, and for quantifying protospacer sequences and UMI abundance in recombination quantification).

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296] Dataset S5

[0297] FISH probe sequences for barcoding, reporter genes, and phenotypic imaging. This dataset includes separate lists for the following oligonucleotide probes:

[0298] Primary amplification probes for barcode imaging

[0299] Secondary amplification probes for barcode imaging

[0300] FISH probes for RNA containing polyadenylation

[0301] FISH probe for MALAT1

[0302] FISH probes for 7SK

[0303] FISH probes for MRP

[0304] FISH probes for pre-ribosomes

[0305] FISH probe for U2 snRNA

[0306] Oligonucleotide probes for FISH of reporter genes Puro-T2A-mCherry or mCherry-luciferase and SON antibody-linked

[0307] Readout probes for all of the above targets

[0308] Some probes are modified and the modifications are indicated in the probe sequence.

[0309]

[0310]

[0311]

[0312] Although several embodiments of the present invention have been described and illustrated herein, a person of ordinary skill in the art will readily appreciate various other means and / or structures for performing the functions described herein and / or obtaining the results and / or one or more advantages described herein, and each of these changes and / or modifications is considered to be within the scope of the present invention. More generally, it will be readily understood by those skilled in the art that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on one or more specific applications for which the teachings of the present invention are used. Those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments of the present invention described herein by using no more than routine experiments. Therefore, it should be understood that the foregoing embodiments are given by way of example only, and within the scope of the appended claims and their equivalents, the present invention may be implemented in a manner different from that specifically described and claimed. The present invention relates to each individual feature, system, article, material, kit, and / or method described herein. In addition, if these features, systems, articles, materials, kits, and / or methods do not contradict each other, any combination of two or more such features, systems, articles, materials, kits, and / or methods is also included within the scope of the present invention.

[0313] In the event that this specification and the documents incorporated by reference include conflicting and / or inconsistent disclosures, this specification shall prevail. If two or more documents incorporated by reference include conflicting and / or inconsistent disclosures, the document with the later effective date shall prevail.

[0314] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0315] The indefinite articles "a" and "an" as used herein in the specification and claims should be understood to mean "at least one" unless clearly indicated to the contrary.

[0316] As used herein in the specification and claims, the phrase "and / or" should be understood to mean "either or both" of the connected elements, i.e., the elements are present in combination in some cases and separately in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the connected elements. In addition to the elements explicitly identified by the "and / or" clause, other elements may optionally be present, whether related or unrelated to those elements explicitly identified. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with an open-ended phrase such as "comprising", may refer to A only (optionally including elements other than B) in one embodiment; may refer to B only (optionally including elements other than A) in another embodiment; may refer to A and B (optionally including other elements) in another embodiment; and so on.

[0317] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be understood to be inclusive, i.e., including at least one, but also including more than one of a plurality of elements or a list of elements, and optionally, including additional unlisted items. Only terms that clearly indicate the contrary, such as "only one of..." or "exactly one of..." or "consisting of..." when used in a claim will refer to the inclusion of exactly one element of a plurality of elements or a list of elements. Generally, the term "or" as used herein should be interpreted as indicating an exclusive choice (i.e., "one or the other but not both") only when it is preceded by an exclusive term such as "either," "one of...", "only one of..." or "exactly one of..."

[0318] As used herein in the specification and claims, the phrase "at least one" with respect to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each element explicitly listed in the list of elements and does not exclude any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than the elements explicitly identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to those explicitly identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") may refer in one embodiment to at least one (optionally including more than one) A without B present (and optionally including elements other than B); in another embodiment to at least one (optionally including more than one) B without A present (and optionally including elements other than A); in another embodiment to at least one (optionally including more than one) A and at least one (optionally including more than one) B (and optionally including other elements); and so on.

[0319] When the word "about" is used herein to refer to a number, it should be understood that another embodiment of the present invention includes the number not modified by the word "about."

[0320] It should also be understood that, unless explicitly stated to the contrary, in any method claimed herein that includes more than one step or act, the order of the steps or acts of the method are not necessarily limited to the order in which the steps or acts of the method are recited.

[0321] In the claims, as well as in the foregoing specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "having," "consisting of," and the like are to be construed as open-ended, i.e., meaning including, but not limited to, "consisting of." Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in Section 2111.03 of the U.S. Patent Office Manual of Patent Examining Procedures.

Claims

1. A method comprising: (a) introducing DNA into a plurality of cells, wherein the DNA comprises: a guide portion comprising a recognition sequence, a reporter gene portion, and an identification portion comprising a reading sequence; (b) determining the location of RNA molecules expressed by the reporter gene portion of the introduced DNA in the plurality of cells by determining the reporter gene portion; (c) determining a read sequence on the RNA molecule expressed by the introduced DNA comprising the reporter gene portion and the marker portion in the plurality of cells by exposing the cells to a read probe capable of binding to the read sequence; (d) co-localizing the binding of the readout probe with the position of the RNA molecule expressed by the reporter gene portion of the introduced DNA; (e) repeating (b), (c) and (d) multiple times using different read sequences; and (f) forming a codeword corresponding to the binding of the co-localized read probe, wherein a bit value of the codeword is based on the binding of the read probe to the read sequence.

2. The method of claim 1, further comprising identifying a guide portion of a single cell based on the measured codeword.

3. The method of any one of claims 1 or 2, wherein the recognition sequence recognizes a DNA sequence.

4. The method of any one of claims 1-3, wherein the recognition sequence recognizes an RNA sequence.

5. The method of any one of claims 1-4, wherein the introduced DNA is derived from a nucleic acid library. The method of claim 5 , wherein the library is generated by mixed cloning.

7. The method of any one of claims 1-6, wherein the identity of the associated pair of the guide moiety and the marker moiety on the DNA is determined by sequencing.

8. The method of any one of claims 1-7, wherein the introduced DNA allows the association of the guide moiety and the marker moiety to occur.

9. The method of any one of claims 1-8, wherein each read sequence represents a value of a position within a codeword.

10. The method of any one of claims 1-9, wherein the read sequences are determined sequentially.

11. The method of any one of claims 1-10, wherein the guide portion further comprises a Cas protein binding sequence.

12. The method of any one of claims 1-11, wherein the guide portion allows the Cas protein to target DNA or RNA to interfere with the sequence or expression of a gene.

13. The method of any one of claims 1-12, wherein the reporter gene portion encodes a protein that can be detected by fluorescence.

14. The method of any one of claims 1-13, wherein the reporter gene portion encodes a fluorescent protein.

15. The method of any one of claims 1-14, wherein the reporter gene portion encodes luciferase.

16. The method of any one of claims 1-15, wherein the reporter gene portion encodes a protein that can be detected by immunoprecipitation.

17. The method of any one of claims 1-16, wherein the reporter gene portion encodes a protein that can be detected by immunofluorescence.

18. The method of any one of claims 1-17, wherein the reporter gene portion encodes a Myc tag.

19. The method of any one of claims 1-18, wherein the reporter gene portion encodes an HA tag.

20. The method of any one of claims 1-19, wherein the reporter gene moiety comprises a reporter gene.

21. The method of any one of claims 1-20, wherein the identification moiety is present in the 3'UTR of the reporter gene.

22. The method of any one of claims 1-21, wherein the reporter gene portion comprises a first promoter.

23. The method of claim 22, wherein the promoter is a promoter that drives transcription.

24. The method of any one of claims 22 or 23, wherein the promoter comprises a CMV promoter.

25. The method of any one of claims 22-24, wherein the recognition sequence comprises a second promoter separate from the first promoter.

26. The method of any one of claims 1-25, wherein the recognition sequence and the reporter gene portion are separated by at least 1000 bases.

27. The method of any one of claims 1-26, wherein the recognition sequence and the reporter gene portion are separated by at least 100 bases within the DNA.

28. The method of any one of claims 1-27, comprising determining the one or more read sequences using fluorescence.

29. The method of any one of claims 1-28, comprising determining the location of RNA comprising the reporter gene portion and the marker portion using smFISH targeted to the reporter gene portion.

30. The method of any one of claims 1-29, comprising introducing the DNA into the plurality of cells using a virus.

31. The method of claim 30, wherein the virus is a lentivirus.

32. The method of claim 31, wherein the guide moiety and the tag moiety are positioned immediately 3' to a polypurine band sequence within the lentivirus.

33. The method of claim 32, wherein the guide portion replicates within the 5' region of the lentivirus.

34. The method of any one of claims 1-33, wherein introducing the DNA into the plurality of cells comprises electroporating the DNA into the plurality of cells.

35. The method of any one of claims 1-34, wherein the cells comprise cells in a tissue.

36. The method of any one of claims 1-35, comprising introducing the DNA into the plurality of cells such that at least 50% of the cells contain no more than one type of introduced DNA.

37. The method of any one of claims 1-36, comprising introducing the DNA into the plurality of cells such that at least 90% of the cells contain no more than one type of introduced DNA.

38. The method of any one of claims 1-37, comprising introducing the DNA into the genome of the cell.

39. The method of any one of claims 1-38, wherein the DNA further comprises a promoter.

40. The method of any one of claims 1-39, wherein the read sequence defines a binary space of the number of bits of the codeword.

41. The method of any one of claims 1-40, wherein the read sequence defines a ternary space of the number of bits of the codeword.

42. The method of any one of claims 1-41, wherein determining the one or more read sequences comprises: For each bit number of the codeword, a readout probe corresponding to the bit number of the codeword is applied to the plurality of cells.

43. The method of claim 42, comprising sequentially applying and removing each of the readout probes to the plurality of cells.

44. The method of any one of claims 1-43, wherein for at least some of the formed codewords, the codewords are matched with valid codewords, wherein: If no match is found, the codeword is discarded or error correction is applied to the codeword to form a valid codeword.

45. The method of claim 44, wherein the value of the number of bits of the codeword defines a set of potential codewords.

46. ​​The method of claim 45, wherein the set of potential codewords includes at least 10 2 A single sequence.

47. The method of claim 46, wherein the set of potential codewords includes at least 10 3 A single sequence.

48. The method of any one of claims 46 or 47, wherein the set of potential codewords comprises at least 10 4 A single sequence.

49. The method of any one of claims 46-48, wherein the set of potential codewords includes at least 10 5 A single sequence.

50. The method of any one of claims 46-49, wherein the set of potential codewords includes at least 10 6 A single sequence.

51. The method of any one of claims 44-50, wherein a portion of the potential codewords are valid codewords.

52. The method of claim 51, comprising comparing the measured codeword to the valid codeword to determine an error in the codeword measurement.

53. The method of any one of claims 44-52, wherein the valid codewords are a randomly selected subset of possible codewords.

54. The method of claim 53, wherein less than 10% of possible codewords are valid codewords.

55. The method of any one of claims 53 or 54, wherein less than 5% of possible codewords are valid codewords.

56. The method of any of claims 53-55, wherein less than 1% of possible codewords are valid codewords.

57. The method of any one of claims 53-56, wherein less than 0.5% of possible codewords are valid codewords.

58. The method of any one of claims 1-57, wherein the identification portion comprises N variable portions, N is at least 3, each variable portion having at least two possibilities.

59. The method of claim 58, wherein N is at least 5.

60. The method of any one of claims 58 or 59, wherein N is at least 10.

61. The method of any one of claims 58-60, wherein N is at least 15.

62. The method of any one of claims 58-61, wherein N is at least 20.

63. The method of any one of claims 1-62, wherein the variable portions within the identification portion each have the same length.

64. The method of any one of claims 1-63, wherein the variable portions within the identification portion each have a length of 5-50 nt.

65. The method of any one of claims 1-64, wherein the variable portions within the identification portion each have a length of 15-25 nt.

66. The method of any one of claims 1-65, wherein the identification portion comprises an error detection code.

67. The method of any one of claims 1-66, wherein the identification portion comprises an error correction code.

68. The method of any one of claims 66 or 67, wherein the error detection or correction code comprises a Hamming code.

69. The method of any one of claims 66-68, wherein the error detection code or error correction code comprises an extended Hamming code.

70. The method of any one of claims 66-69, wherein the error detection or correction code comprises a Reed-Solomon code.

71. The method of any one of claims 66-70, wherein the error detection code or error correction code is not the same for all members of the code.

72. The method of any one of claims 1-71, wherein the nucleic acid probe comprises at least 8 possible read sequences.

73. The method of any one of claims 1-72, wherein the nucleic acid probe comprises at least 16 possible read sequences.

74. The method of any one of claims 1-73, wherein the nucleic acid probe comprises no more than 32 possible read sequences.

75. The method of any one of claims 1-74, wherein the nucleic acid probe comprises at least 32 possible read sequences.

76. A method comprising: introducing DNA into a plurality of cells, the DNA comprising: a guide portion comprising a recognition sequence, a reporter gene portion, and an identification portion comprising a reader sequence; determining the location of RNA molecules expressed by the reporter gene portion of the introduced DNA within the plurality of cells by determining the reporter gene portion; determining the read sequence within the plurality of cells by exposing the cells to a plurality of read probes each capable of binding to the read sequence, colocalizing the binding of the readout probe with the position of the RNA molecule expressed by the reporter gene portion of the introduced DNA; and A codeword is formed corresponding to the binding of the co-localized read probes, wherein a bit value of the codeword is based on the binding of the read probe to the read sequence.

77. A method comprising: introducing DNA into a plurality of cells using a lentivirus, wherein the DNA comprises: a guide portion comprising a recognition sequence, a reporter gene portion, and an identification portion comprising a read sequence; determining a phenotype of the plurality of cells; and determining the genotype of the plurality of cells; as well as A correspondence between the genotype and the phenotype is determined.

78. The method of claim 77, wherein determining the phenotype comprises determining a property of a cell.

79. The method of any one of claims 77 or 78, wherein determining the phenotype comprises determining the morphology of the cell structure.

80. The method of claim 79, wherein the morphology is a whole-cell morphology.

81. The method of any one of claims 79 or 80, wherein the morphology is a subcompartmental morphology.

82. The method of any one of claims 77-81, wherein determining the phenotype comprises determining the morphology of more than one cellular structure.

83. The method of any one of claims 77-82, wherein determining the phenotype comprises determining a protein using immunofluorescence.

84. The method of any one of claims 77-83, wherein determining the phenotype comprises determining a protein using fluorescence.

85. The method of any one of claims 77-84, wherein determining the phenotype comprises determining the protein using a fluorescent protein.

86. The method of any one of claims 77-85, wherein determining the phenotype comprises determining a protein using an organic dye.

87. The method of any one of claims 77-86, wherein determining the phenotype comprises determining cellular kinetic behavior.

88. The method of any one of claims 77-87, wherein determining the phenotype comprises determining cell-cell interactions.

89. The method of any one of claims 77-88, wherein determining the phenotype comprises determining a cell state.

90. The method of any one of claims 77-89, wherein determining the phenotype comprises determining a cellular RNA or multiple cellular RNAs using smFISH.

91. The method of any one of claims 77-90, wherein determining the phenotype comprises determining a gene expression profile using multiplex FISH.

92. The method of any one of claims 77-91, wherein determining the phenotype comprises determining a gene expression profile using MERFISH.

93. The method of any one of claims 77-92, wherein determining a phenotype comprises spatially determining an RNA or multiple RNAs.

94. The method of any one of claims 77-93, wherein determining a phenotype comprises determining at least a portion of a proteome.

95. The method of any one of claims 77-94, wherein determining the phenotype comprises determining at least a portion of a chromosome using DNA FISH.

96. The method of any one of claims 77-95, wherein determining the phenotype comprises determining at least a portion of a chromosome using multiplex DNA FISH.

97. The method of any one of claims 77-96, wherein determining the phenotype comprises determining at least a portion of a chromosome using CASFISH.

98. The method of any one of claims 77-97, wherein determining the phenotype comprises determining protein modified cells using immunofluorescence.

99. The method of any one of claims 77-98, wherein determining the phenotype comprises determining the interaction of the protein with RNA or DNA.

100. The method of any one of claims 77-99, wherein determining a phenotype comprises determining an epigenetic modification.

101. The method of any one of claims 77-100, wherein determining the phenotype comprises determining cell growth.

102. The method of any one of claims 77-101, wherein determining the phenotype comprises determining a change in cell growth.

103. The method of any one of claims 77-102, wherein determining the phenotype and determining the genotype use common imaging techniques.

104. The method of any one of claims 77-103, wherein determining the phenotype and determining the genotype use different imaging techniques.

105. The method of any one of claims 103 or 104, wherein the imaging technique has a resolution exceeding 300 nm.

106. The method of any one of claims 103-105, wherein determining the phenotype uses multi-color fluorescence imaging.

107. The method of any one of claims 103-106, wherein determining the phenotype uses confocal imaging.

108. The method of any one of claims 103-107, wherein determining the phenotype uses TIRF imaging.

109. The method of any one of claims 103-108, wherein determining the phenotype uses two-photon imaging.

110. The method of any one of claims 103-109, wherein determining the phenotype uses STORM.

111. The method of any one of claims 103-110, wherein determining the phenotype uses super-resolution technology.

112. The method of claim 111, wherein the super-resolution technique is PALM, FPALM, STED, SIM and / or RESOLFT.

113. The method of any one of claims 77-112, wherein determining the genotype comprises determining the sequence of an identification portion of the introduced DNA.

114. The method of any one of claims 77-113, wherein determining the genotype comprises determining the genotype using smFISH.

115. The method of any one of claims 77-114, wherein determining the genotype comprises determining the genotype using multiplex FISH.

116. The method of any one of claims 77-115, wherein determining the genotype comprises determining the genotype using MERFISH.

117. The method of any one of claims 77-116, wherein determining the genotype comprises determining the genotype using in situ hybridization.

118. The method of any one of claims 77-117, wherein determining the genotype comprises determining the genotype using serial FISH.

119. The method of any one of claims 77-118, wherein determining the genotype comprises determining the genotype using CASFISH.

120. The method of any one of claims 77-119, wherein determining the genotype comprises determining the genotype using in situ sequencing.

121. A method comprising: introducing a nucleic acid into a plurality of cells, wherein the nucleic acid comprises: a guide portion comprising a recognition sequence, a reporter gene portion, and an identification portion comprising a reader sequence; imaging the plurality of cells, wherein the cells exhibit an imageable phenotypic difference due to expression of the guide moiety; and A plurality of images of the plurality of cells are acquired, wherein the cell images exhibit differences due to differences in the labeled portions of the nucleic acid within the cells.

122. The method of claim 121, wherein the guide portion introduced into the plurality of cells is identified based on a correspondence between the guide portion and the identification portion.

123. The method of any one of claims 121 or 122, wherein cells of different phenotypes have a different appearance when imaged.

124. The method of any one of claims 121-123, wherein cells with different phenotypes have different fluorescence when imaged.

125. The method of any one of claims 121-124, wherein imaging the plurality of cells comprises acquiring images of the plurality of cells using a single imaging modality.

126. The method of any one of claims 121-125, wherein imaging the plurality of cells comprises acquiring images of the plurality of cells using a plurality of imaging modalities.

127. The method of any one of claims 121-126, wherein imaging the plurality of cells comprises acquiring a single image.

128. The method of any one of claims 121-127, wherein imaging the plurality of cells comprises acquiring a plurality of images.

129. The method of any one of claims 121-128, wherein imaging the plurality of cells comprises imaging the plurality of cells using smFISH.

130. The method of any one of claims 121-129, wherein imaging the plurality of cells comprises imaging the plurality of cells using MERFISH.

131. The method of any one of claims 121-130, further comprising determining the morphology of the cell.

132. The method of claim 131, comprising determining morphological changes in the cells over time.

133. The method of any one of claims 131 or 132, comprising determining a morphological change in an organelle of the cell.

134. The method of any one of claims 131-133, comprising determining morphological changes during cell growth.

135. A method comprising: introducing DNA into a plurality of cells using a lentivirus, wherein the DNA comprises: a guide portion comprising a recognition sequence and an identification portion comprising a read sequence; determining a phenotype of the plurality of cells; determining the genotype of the plurality of cells; and Determine the correspondence between genotype and phenotype.

136. The method of claim 135, wherein determining the phenotype comprises determining a property of a cell.

137. The method of any one of claims 135 or 136, wherein determining the phenotype comprises determining the morphology of the cell structure.

138. The method of claim 137, wherein the morphology is a whole-cell morphology.

139. The method of any one of claims 137 or 138, wherein the morphology is a subcompartmental morphology.

140. The method of any one of claims 137-139, wherein determining the phenotype comprises determining the morphology of more than one cellular structure.

141. The method of any one of claims 135-140, wherein determining the phenotype comprises determining a protein using immunofluorescence.

142. The method of any one of claims 135-141, wherein determining the phenotype comprises determining a protein using fluorescence.

143. The method of any one of claims 135-142, wherein determining the phenotype comprises determining the protein using a fluorescent protein.

144. The method of any one of claims 135-143, wherein determining the phenotype comprises determining a protein using an organic dye.

145. The method of any one of claims 135-144, wherein determining the phenotype comprises determining cellular kinetic behavior.

146. The method of any one of claims 135-145, wherein determining the phenotype comprises determining cell-cell interactions.

147. The method of any one of claims 135-146, wherein determining the phenotype comprises determining a cell state.

148. The method of any one of claims 135-147, wherein determining the phenotype comprises determining one or more RNAs using smFISH.

149. The method of any one of claims 135-148, wherein determining the phenotype comprises determining a gene expression profile using multiplex FISH.

150. The method of any one of claims 135-149, wherein determining the phenotype comprises determining a gene expression profile using MERFISH.

151. The method of any one of claims 135-150, wherein determining a phenotype comprises spatially determining one or more RNAs.

152. The method of any one of claims 135-151, wherein determining a phenotype comprises determining at least a portion of a proteome.

153. The method of any one of claims 135-152, wherein determining the phenotype comprises determining at least a portion of a chromosome using DNA FISH.

154. The method of any one of claims 135-153, wherein determining the phenotype comprises determining at least a portion of a chromosome using multiplex DNA FISH.

155. The method of any one of claims 135-154, wherein determining the phenotype comprises determining at least a portion of a chromosome using CASFISH.

156. The method of any one of claims 135-155, wherein determining the phenotype comprises determining protein modified cells using immunofluorescence.

157. The method of any one of claims 135-156, wherein determining the phenotype comprises determining an interaction of a protein with RNA or DNA.

158. The method of any one of claims 135-157, wherein determining a phenotype comprises determining an epigenetic modification.

159. The method of any one of claims 135-158, wherein determining the phenotype comprises determining cell growth.

160. The method of any one of claims 135-159, wherein determining a phenotype comprises determining a change in cell growth.

161. The method of any one of claims 135-160, wherein determining the phenotype and determining the genotype use common imaging techniques.

162. The method of claim 161, wherein the imaging technique has a resolution greater than 300 nm.

163. The method of any one of claims 135-160, wherein determining the phenotype and determining the genotype use different imaging techniques.

164. The method of claim 163, wherein at least one of the imaging techniques has a resolution greater than 300 nm.

165. The method of any one of claims 135-164, wherein determining the phenotype uses multi-color fluorescence imaging.

166. The method of any of claims 135-165, wherein determining the phenotype uses confocal imaging.

167. The method of any one of claims 135-166, wherein determining the phenotype uses TIRF imaging.

168. The method of any of claims 135-167, wherein determining the phenotype uses two-photon imaging.

169. The method of any one of claims 135-168, wherein determining the phenotype uses STORM.

170. The method of any one of claims 135-169, wherein determining the phenotype uses super-resolution technology.

171. The method of any one of claims 135-170, wherein the super-resolution technique is PALM, FPALM, STED, SIM and / or RESOLFT.

172. The method of any one of claims 135-171, wherein determining the genotype comprises determining the sequence of an identification portion of the introduced DNA.

173. The method of any one of claims 135-172, wherein determining the genotype comprises determining the genotype using smFISH.

174. The method of any one of claims 135-173, wherein determining the genotype comprises determining the genotype using multiplex FISH.

175. The method of any one of claims 135-174, wherein determining the genotype comprises determining the genotype using MERFISH.

176. The method of any one of claims 135-175, wherein determining the genotype comprises determining the genotype using in situ hybridization.

177. The method of any one of claims 135-176, wherein determining the genotype comprises determining the genotype using serial FISH.

178. The method of any one of claims 135-177, wherein determining the genotype comprises determining the genotype using CASFISH.

179. The method of any one of claims 135-178, wherein determining the genotype comprises determining the genotype using in situ sequencing.

Images