Systems and methods for screening of biological components

Abstract

Described herein are methods and systems for utilizing the formation and degradation of hydrogel chambers to analyze biological components comprising guide RNA barcodes associated with one or more genetic modifications. A method for analyzing a biological component can include introducing the biological component into a fluidic device; in the fluidic device, encapsulating the biological component in a hydrogel chamber, wherein the biological component comprises a guide ribonucleic acid (RNA) molecule associated with a genetic modification of the biological component; and releasing the guide RNA molecule from the biological component.

Description

SYSTEMS AND METHODS FOR SCREENING OF BIOLOGICAL COMPONENTSCROSS-REFERENCE

[0001] This application claims the benefit of French (FR) Patent Application No. 2404680, filed May 3, 2024, which application is incorporated herein by reference in its entirety for all purposes.BACKGROUND

[0002] CRISPR screens have improved our understanding of genetic functionality and interactions, bringing forth a powerful tool for comprehensive genetic studies. The emergence of single-cell resolution in CRISPR screens has further amplified this value, offering a lens through which to view the complex dynamics of genetic networks. This technique allows observation of the effects of genetic alterations at the individual cell level, which provides a more nuanced understanding of gene functions and their context-dependent behaviors. As a result, single-cell resolution enhances the precision and depth of our genetic analyses, making it a valuable tool in the field of genomics.SUMMARY

[0003] Described herein is a method of achieving single-cell resolution for CRISPR screening by combining 1) transcriptome profiling, 2) guide-RNA sequencing, and 3) functional readouts of individual cells at scale using targeted formation and degradation of hydrogel chambers on a fluidic device.

[0004] In an aspect, provided herein is a method for analyzing a biological component, the method comprising: (a) introducing the biological component into a fluidic device; (b) in the fluidic device, encapsulating the biological component in a hydrogel chamber, wherein the biological component comprises a guide RNA associated with a genetic modification of the biological component; and (c) releasing the guide RNA from the biological component.

[0005] In some cases, the fluidic device comprises one or more capture probes. In some cases, subsequent to (c), the one or more capture probes capture the guide RNA released from the biological component. In some cases, the one or more capture probes each comprise a spatial location tag corresponding to a unique location of the capture probe on the fluidic device. In some cases, fluidic device comprises one or more arrays located on a surface of the fluidic device, andeach array comprises one or more reaction sites. A reaction site of the one or more reaction sites can comprise the spatial location tag. The spatial location tag can comprise: (a) a first oligonucleotide sequence unique to a spatial location of the reaction site within the array; and (b) a second oligonucleotide unique to the array of the one or more arrays. In some cases, unique location comprises at least a portion of the reaction site.

[0006] In some cases, a capture probe of the one or more capture probes comprises an anti- polyA probe configured to couple with the guide RNA. In some cases, the guide RNA comprises a single guide RNA (sgRNA). In some cases, the guide RNA comprises an exogenous messenger RNA (mRNA), and the exogenous mRNA comprises a single guide RNA (sgRNA) sequence. In some cases, the guide RNA comprises an exogenous messenger RNA (mRNA), and the exogenous mRNA comprises a barcode encoding a single guide RNA (sgRNA) sequence.

[0007] In some cases, the biological component further comprises a messenger RNA (mRNA), and the mRNA is released from the biological component in (c). In some cases, the method further comprises subsequent to (c), generating a cDNA guide barcode based on the guide RNA and generating a cDNA segment based on the mRNA and then sequencing the cDNA guide barcode and the cDNA segment. In some cases, the method further comprises eluting the cDNA guide barcode and the cDNA segment from the fluidic device to form an elution mixture and amplifying the cDNA guide barcode and the cDNA segment to form an amplification mixture using a whole transcriptome primer configured to initiate amplification with the cDNA segment and using a gene specific primer configured to initiate amplification with the cDNA guide barcode.

[0008] In some cases, the method further comprises correlating a result of one or more assays performed on the biological component with the cDNA guide barcode of the biological component. In some cases, the method further comprises introducing additional biological components into the fluidic device, and the biological component is at least partially selected from the additional biological components based on a result of one or more assays performed on the biological component and the additional biological components. In some cases, the performing of the one or more assays occurs prior to (b). In some cases, the performing of the one or more assays occurs subsequent to (b). In some cases, the method further comprises, based on the result of the one or more assays, (i) degrading the hydrogel chamber encapsulating the biological component or (ii) degrading an additional chamber encapsulating an additional biological component. In some cases, the one or more assays comprises a fluorescence assay. In some cases, the one or moreassays comprise a secretion assay. In some cases, the secretion assay comprises measuring one or more secretions of the biological component and the additional biological components. In some cases, the one or more secretions comprise a cytokine. In some cases, the method further comprises degrading the hydrogel chamber encapsulating the biological component when a signal associated with the cytokine is below a threshold value. In some cases, the method further comprises, subsequent to (b), removing additional biological components having cytokine signals below a threshold value.

[0009] In some cases, the biological component comprises a T cell. In some cases, the one or more assays comprise measuring one or more surface markers of the one or more biological components. In some cases, the one or more surface markers comprise CD3, CD4, CD8, CD19, CD25, CD45, or CD56, or any combination thereof.

[0010] In some cases, the one or more assays comprise measuring an interaction between the biological component and an additional biological component. In some cases, the additional biological component is encapsulated in the hydrogel chamber with the biological component. In some cases, the interaction comprises a killing of the biological component by the additional biological component. In some cases, the biological component comprises a T cell. In some cases, the biological component comprises a CD8+ T cell. In some cases, the biological component comprises a CAR-T cell. In some cases, the interaction comprises a killing of the additional biological component by the biological component. In some cases, the additional biological component comprises a CD8+ T cell. In some cases, the additional biological component comprises a CAR-T cell.

[0011] In some cases, the interaction comprises a proliferation of the biological component or the additional biological component. In some cases, the interaction comprises a physical contact between the biological component and the additional biological component.

[0012] In some cases, the biological component comprises a neuron. In some cases, the method further comprises measuring Ca2+ signaling of the neuron. In some cases, the method further comprises measuring ATP release of the neuron. In some cases, the method further comprises screening the neuron for deoxyribonucleic acid (DNA) double strand breaks. In some cases, the method further comprises determining a morphological classification of the neuron. In some cases, the morphological classification comprises a unipolar neuron. In some cases, the morphological classification comprises a bipolar neuron. In some cases, the morphological classificationcomprises a multipolar neuron. In some cases, the morphological classification comprises a pseudo-unipolar neuron. In some cases, the biological component comprises a dendritic cell. In some cases, the method further comprises measuring an antigen presentation of the dendritic cell. In some cases, the method further comprises measuring activation of a T cell by the dendritic cell.

[0013] In some cases, the genetic modification comprises a CRISPR-modification. In some cases, the biological component expresses Cas9 or a variant thereof.

[0014] In some cases, the method further comprises imaging the biological component. In some cases, the method further comprises imaging the fluidic device. In some cases, the method further comprises introducing one or more polymer precursors into the fluidic device. In some cases, the one or more polymer precursors comprise a crosslinker, a porogen, and a photoinitiator. In some cases, the one or more polymer precursors are introduced into the fluidic device at a same time as the introducing of the biological component in (a).

[0015] In another aspect, provided herein is a fluidic device for analyzing a biological component, comprising: a hydrogel chamber encapsulating the biological component, wherein the biological component comprises a guide RNA associated with a genetic modification of the biological component; and one or more capture probes configured to capture the guide RNA upon release from the biological component.

[0016] In some cases, the one or more capture probes each comprise a spatial location tag corresponding to a unique location of the capture probe on the fluidic device. In some cases, the fluidic device comprises one or more arrays on a surface of the fluidic device, and each array comprises one or more reaction sites. A reaction site of the one or more reaction sites can comprise the spatial location tag. In some cases, the spatial location tag comprises: (a) a first oligonucleotide sequence unique to a spatial location of the reaction site within the array; and (b) a second oligonucleotide unique to the array of the one or more arrays. In some cases, the unique location comprises at least a portion of the reaction site. In some cases, a capture probe of the one or more capture probes comprises an anti-polyA probe configured to couple with the guide RNA. In some cases, the guide RNA comprises a single guide RNA (sgRNA). In some cases, the guide RNA comprises an exogenous messenger RNA (mRNA), and the exogenous mRNA comprises a single guide RNA (sgRNA) sequence. In some cases, the guide RNA comprises an exogenous messenger RNA (mRNA), and the exogenous mRNA comprises a barcode encoding a single guide RNA (sgRNA) sequence. In some cases, biological component further comprises a messenger RNA(mRNA), and the one or more capture probes are configured to capture the mRNA upon release from the biological component.

[0017] In some cases, the fluidic device further comprises additional biological components, and the biological component is at least partially selected from the additional biological components based on a result of one or more assays performed on the biological component and the additional biological components. In some cases, the one or more assays comprises a fluorescence assay. In some cases, the one or more assays comprise a secretion assay. In some cases, the secretion assay comprises measuring one or more secretions of the biological component and the additional biological components. In some cases, the one or more secretions comprise a cytokine. In some cases, the biological component comprises a T cell. In some cases, the one or more assays comprise measuring one or more surface markers of the one or more biological components. In some cases, the one or more surface markers comprise CD3, CD4, CD8, CD19, CD25, CD45, or CD56, or any combination thereof.

[0018] In some cases, the one or more assays comprise measuring an interaction between the biological component and an additional biological component. In some cases, the additional biological component is encapsulated in the hydrogel chamber with the biological component. In some cases, the interaction comprises a killing of the biological component by the additional biological component. In some cases, the biological component comprises a T cell. In some cases, the biological component comprises a CD8+ T cell. In some cases, the biological component comprises a CAR-T cell. In some cases, the interaction comprises a killing of the additional biological component by the biological component. In some cases, the additional biological component comprises a CD8+ T cell. In some cases, the additional biological component comprises a CAR-T cell. In some cases, the interaction comprises a proliferation of the biological component or the additional biological component. In some cases, the interaction comprises a physical contact between the biological component and the additional biological component.

[0019] In some cases, the biological component comprises a neuron. In some cases, the genetic modification impacts Ca2+ signaling of the neuron. In some cases, the genetic modification impacts ATP release of the neuron. In some cases, the genetic modification impacts deoxyribonucleic acid (DNA) double strand breaks of the neuron. In some cases, the neuron comprises a unipolar neuron. In some cases, the neuron comprises a bipolar neuron. In some cases, the neuron comprises a multipolar neuron. In some cases, the neuron comprises a pseudo-unipolarneuron. In some cases, the biological component comprises a dendritic cell. In some cases, the genetic modification impacts an antigen presentation of the dendritic cell. In some cases, the genetic modification impacts activation of a T cell by the dendritic cell. In some cases, the genetic modification comprises a CRISPR-modification. In some cases, the biological component expresses Cas9 or a variant thereof.

[0020] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, where only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.INCORPORATION BY REFERENCE

[0021] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the systems, devices, and methods described herein will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles described herein are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0023] FIG. 1A depicts a schematic example of a CRISPR screening assay using hydrogel chambers and sequencing based guide identification, in accordance with some embodiments.

[0024] FIG. IB depicts a schematic example of a CRISPR screening assay using hydrogel chambers, a functional readout and sequencing based guide identification, in accordance with some embodiments.

[0025] FIG. 2 depicts a schematic example of library preparation of the guide RNA and the mRNA, in accordance with some embodiments.

[0026] FIG. 3 shows the results of a library preparation by concentration (ng / mL) and product size distribution, in accordance with some embodiments.

[0027] FIG. 4 shows the performance of mRNA sequencing with and without hydrogel chambers (i.e. cages), in accordance with some embodiments.

[0028] FIG. 5 shows a barcoded flow cell array, in accordance with some embodiments.

[0029] FIG. 6 shows fluorescent based identification of cells on a portion of a channel disposed in a fluidic device, in accordance with some embodiments.

[0030] FIG. 7 shows a distribution of cells based on fluorescent intensity for fluorescent based cell identification of cells on a portion of a channel disposed in a fluidic device, in accordance with some embodiments.

[0031] FIG. 8 shows density plots of transduced cells based on fluorescent intensity, in accordance with some embodiments.

[0032] FIG. 9 shows density plots of unique molecular identifiers (UMIs) with and without hydrogel chambers (i.e. cages), in accordance with some embodiments.

[0033] FIG. 10 shows density plots of unique molecular identifiers (UMIs) grouped by fluorescent based identification of guide RNA, in accordance with some embodiments.

[0034] FIG. 11 shows density plots by object area size between low and high UMI cells, in accordance with some embodiments.

[0035] FIG. 12 shows density plots by barcode-hydrogel chamber (i.e. cage) overlap percentage of low and high UMI cells, in accordance with some embodiments.

[0036] FIG. 13 shows a plot of non-targeting guide RNA UMI counts of all hydrogel chamber caged cells and hydrogel chamber caged cells with greater than 13% barcode overlap, in accordance with some embodiments.

[0037] FIG. 14 shows a plot of targeting guide RNA UMI counts of all hydrogel chamber caged cells and hydrogel chamber caged cells with greater than 13% barcode overlap, in accordance with some embodiments.

[0038] FIG. 15 shows a schematic illustration of a portion of a channel disposed in a fluidic device, according to some embodiments, in accordance with some embodiments.

[0039] FIG. 16 shows a portion of a system as provided herein including an energy source, according to some embodiments, in accordance with some embodiments.

[0040] FIG. 17 shows a computer system that is programmed or otherwise configured to implement methods provided herein.

[0041] FIG. 18 is a plot of cell fluorescence intensities, wherein two images of chamber- enclosed cells are displayed at the top of the plot.

[0042] FIG. 19A is a plot of gRNA reads from individual chambers in a channel of a fluidic device.

[0043] FIG. 19B is a plot of gRNA reads from individual chambers in a channel of a fluidic device.

[0044] FIG. 19C is a plot of gRNA reads from individual chambers in a channel of a fluidic device.

[0045] FIG. 19D is a plot of gRNA reads from individual chambers in a channel of a fluidic device.

[0046] FIG. 20 is a plot that displays the difference in sequencing counts for two gRNAs that were captured and measured in individual chambers within a fluidic device.

[0047] FIG. 21 is an illustration of DNA that encodes two guide RNA molecules that are shown in this figure.

[0048] FIG. 22 is an illustration of a gRNA molecule hybridized to a nucleic acid capture probe.DETAILED DESCRIPTIONIntroduction

[0049] Aspects of the present disclosure provide methods for detecting guide RNA from individual cells. Such methods may utilize photopolymerization to generate polymeric chambers enclosing cells of interest. The cells can be chemically and optically analyzed while they are enclosed within the polymeric chambers. Genetic modifications of the cells can then be determined by capturing guide RNA from the cells on spatially barcoded capture probes, and then extending and sequencing the capture probes. Capture probe sequence reads can be associated with individual polymeric chambers (and thus with a cell or cells enclosed within a particular chambers). In this way, thousands of cells can be simultaneously analyzed, and characteristics of these cells can be associated with individual genetic modifications.

[0050] A polymer matrix (e.g., a hydrogel matrix) can be formed adjacent to or around at least of portion of one or more biological components in a fluidic device to isolate selected biological components. A hydrogel matrix may be selectively generated to surround a component. One or more hydrogel or polymer matrix walls can be used to physically separate one or more biological components from one another. Upon degradation of the hydrogel or polymer matrix walls, two or more biological components can interact. These interactions can be monitored and analyzed.

[0051] In some cases, the methods described herein encompass the incorporation of an analyte and one or more biological materials into a hydrogel chamber within the device. In some cases, the analyte and a biological material are initially physically separated by a hydrogel polymer wall. In some cases, degradation of the polymer wall results in an interaction between the analyte and biological material. The interaction can be monitored and detected. In some cases, imaging of the fluidic device or the hydrogel chamber can be used to determine if the analyte has effects on the biological material (or vice versa). The systems and methods described herein can allow for a detailed and targeted approach to analyte analysis, offering an understanding of the interaction between analytes and biological materials in a controlled environment.

[0052] Provided herein is a method for analyzing a biological component, the method comprising: introducing the biological component into a fluidic device. In some embodiments the method further comprises encapsulating the biological component in a hydrogel chamber. In some embodiments the biological component comprises (i) a guide RNA barcode associated with a genetic modification of the biological component and (ii) messenger RNA (mRNA). In some embodiments the method further comprises releasing the guide RNA barcode and mRNA from the biological component.

[0053] In order to compartmentalize individual components of a biological sample, a polymer matrix (e.g., a hydrogel matrix) can be formed adjacent to or around at least of portion of an individual component in a fluidic device. The hydrogel matrix may be selectively generated to surround a component after the system detects the component or hydrogel matrices can be generated according to a predefined pattern in a fluidic device. The hydrogel matrix may allow reagents and smaller entities to pass while retaining the individual component of the biological sample in place. Because one or more individual components can be localized within a fluidicdevice (e.g., encapsulated) and the localized components be exposed to one or more reagents and / or washing solutions during and / or in between analyses, multiple assays can be performed within the compartments (e.g., simultaneously, substantially simultaneously, serially, etc.).

[0054] Different assays may be performed in different locations of the fluidic device, for example, to test effects of different treatment conditions. By having two or more components within a compartment, interactions between components can be studied as well. The polymer matrix can be degradable “on demand” allowing for controlled localization and release mechanisms. The solutions provided herein can retain spatial information of the components and generate data on a cellular, proteomic, transcriptomic, or genomic level. Since spatial information is retained, the data can be associated (e.g., linked) with phenotypic data. Further, the solutions provided herein can retain spatial information of the components and link data (e.g., phenotypic data) on a cellular, proteomic, transcriptomic, or genomic level.

[0055] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0056] Whenever the term “at least” precedes the first numerical value in a series of two or more numerical values, the term “at least” applies to each of the numerical values in that series of numerical values. For example, at least 1, 2, or 3 is equivalent to at least 1, at least 2, or at least 3.

[0057] Whenever the term “less than” precedes the first numerical value in a series of two or more numerical values, the term “less than” applies to each of the numerical values in that series of numerical values. For example, less than 3, 2, or 1 is equivalent to less than 3, less than 2, or less than 1.

[0058] The terms “coupled to,” “connected to,” and “in communication with,” as used herein, generally refer to any form of interaction between two or more entities, including mechanical, electrical, magnetic, electromagnetic, fluid, biological, and thermal interaction. Two components may be coupled to each other even though they are not in direct contact with each other.

[0059] The terms “polypeptide” and “peptide,” as used interchangeably herein, generally refer to a polymer of amino acids in which an amino acid may be linked to another amino acid by a peptide bond. In some examples, a polypeptide is a protein. The amino acid may be a naturallyoccurring amino acid or a non-naturally occurring amino acid (e.g., an amino acid analogue). The polypeptide can be linear or branched. The polypeptide can include modified amino acids. The polypeptide may be interrupted by non-amino acids. A polypeptide can occur as a single chain or an associated chain. The polypeptide may include a plurality of amino acids. The polypeptide may have a secondary and tertiary structure (e.g., the polypeptide may be a protein). In some examples, the polypeptide can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 1,000, 10,000, or more amino acids. The polypeptide may be a fragment of a larger polymer. In some examples, the polypeptide can be a fragment of a larger polypeptide, such as a fragment of a protein.

[0060] The term “amino acid,” as used herein, generally refers to a naturally occurring or non- naturally occurring amino acid (e.g., an amino acid analogue). The non-naturally occurring amino acid may be an engineered or synthesized amino acid.

[0061] The term “sample,” as used herein, generally refers to a chemical or biological sample containing a biological component. The biological component may comprise a cell, a nucleic acid, a microbiome, a protein, a combination of cells, a metabolite, a combination thereof, or any other suitable component of a biological sample. For example, a sample can be a biological sample including one or more cells. For another example, a sample can be a biological sample including one or more polypeptides. The biological sample can be obtained (e.g., extracted or isolated) from or include blood (e.g., whole blood), plasma, serum, urine, saliva, mucosal excretions, sputum, stool, and tears. The biological sample can be a fluid or tissue sample (e.g., skin sample). In some instances, the sample may be derived from a homogenized tissue sample (e.g., brain homogenate, liver homogenate, or kidney homogenate). In certain embodiments, the sample may include a specific type of cell (e.g., a neuronal cell, muscle cell, liver cell, or kidney cell,). The sample may comprise or be acquired from a diseased cell or tissue (e.g., a tumor cell or a necrotic cell), In some embodiments, the sample may include or may be from a disease-associated inclusion (e.g., a plaque, a biofilm, a tumor, or a non-cancerous growth). In certain embodiments, the sample may include or may be obtained from a cell-free bodily fluid, such as whole blood, saliva, or urine. In various embodiments, the sample can include circulating tumor cells. In some cases, the sample may include or may be an environmental sample (e.g., soil, waste, or ambient air), industrial sample (e.g., samples from any industrial processes), or a food sample (e.g., dairy product, vegetable product, or meat product). The sample may be processed prior to loading into amicrofluidic device. For example, the sample may be processed to purify a certain cell type or polypeptide and / or to include reagents.

[0062] As used herein, the term “polymer matrix” generally refers to a phase material (e.g., continuous phase material) that comprises at least one polymer. In some embodiments, the polymer matrix refers to the at least one polymer as well as the interstitial space not occupied by the polymer. A polymer matrix may be composed of one or more types of polymers. A polymer matrix may include linear, branched, and crosslinked polymer units. A polymer matrix may also contain non-polymeric species intercalated within its interstitial spaces not occupied by polymer chains. The intercalated species may be solid, liquid, or gaseous species. For example, the term “polymer matrix” may encompass desiccated hydrogels, hydrated hydrogels, and hydrogels containing glass fibers. A polymer matrix may comprise a polymer precursor, which generally refers to one or more molecules that upon activation can trigger or initiate a polymeric reaction. A polymer precursor can be activated by electrochemical energy, photochemical energy, a photon, magnetic energy, or any other suitable energy. As used herein, the term “polymer precursor” includes monomers (that are polymerized to produce a polymer matrix) and crosslinking compounds, which may include photo-initiators, other compounds necessary or useful for generating polymer matrices, and the like.

[0063] In some embodiments, as used herein, the term “local parameter” means a value of a parameter (such as, pH) in or immediately adjacent to a chamber formed by polymer matrix walls.

[0064] As used herein, the term “on demand” means an operation may be directed to individual, discrete, selected locations (e.g. a spatial location of polymer precursor solution; or a selected polymer matrix chamber). Such selection may be based on manual observation of optical signals or data collected by a detector, or such selection may be based on a computer algorithm operating on optical signals or data collected by a detector. Manual observation of optical signals or data collected by a detector can include either real-time detection or detection at a time period prior to modulating a unit of energy to polymerize polymer precursors or degrading a chamber. For example, a subset of chambers (all formed with photo-degradable polymer matrix walls) may be pre-selected for releasing and removing their contents based on position information and the values of optical signals from an analytical assay carried out in the chambers. The pre-selected chambers may be photo-degraded by selectively projecting a light beam of appropriate wavelength characteristics (for example, with the spatial energy modulating element) to degrade the polymermatrix walls of the pre-selected chambers. In another example, a plurality of chambers may be observed in real-time (e.g. via fluorescent microscopy) for detection of an analyte of interest and one or more chambers of the plurality of chambers is selected, in real-time, upon detection of the analyte of interest, for degradation.

[0065] As used herein, the term “analyte” generally refers to a discrete biological or chemical entity to be measured, detected, and / or distinguished using the methods and systems described herein. In some embodiments, an analyte may be a biological component as described herein.

[0066] The present disclosure provides systems for compartmentalizing or isolating one or more biological components. The system can include a fluidic device containing or including one or more biological components. The fluidic device may contain or include one or more polymer precursors. In some cases, the fluidic device can comprise a first surface configured to couple or receive at least one of the one or more biological components to form a coupled biological component. The systems may also include at least one energy source, wherein the energy source is in communication with the fluidic device. In some embodiments, the energy source may be in optical communication with the fluidic device. In various embodiments, the at least one energy source may form a polymer matrix on or adjacent to at least a portion of the one or more biological components.

[0067] In some cases, a sample may be introduced of provided to the system. In certain cases, the sample may comprise one or more biological components. In various cases, the biological components may be physically separated. In some cases, the biological components may be physically separated but in fluidic communication with one another. In certain cases, the biological components may be in chemical communication with one another. The system may be used for single-cell analysis. In some embodiments, the system may be used for single-cell analysis on a genome level. For example, the system may be used for genome sequencing. For another example, the system may be used for deoxyribonucleic acid (DNA) sequencing. The system may be used for DNA sequencing of cell-free DNA, whole genome sequencing, whole exome sequencing, targeted sequencing, or 16S sequencing. The system may be used for studying DNA tags attached to biomolecules of interest. The biomolecules may comprise proteins, metabolites, etc. In some cases, the DNA may be a nuclear DNA or a mitochondrial DNA. The system may be used for single-cell or bulk analysis on a transcriptome level. For example, the system may be used for ribonucleic acid (RNA) sequencing. For example, the system may be used for 3’ or 5’ geneexpression analysis, immune repertoire study of a cell, or full-length mRNA analysis. In some embodiments, the system may be used for single-cell analysis on a proteome level. The system may be used for functional assay(s) of a biological component. The system may be used for studying surface proteins, secreted proteins, or metabolites of a biological component. In some cases, the system may be used to measure a quality of a biological component. In some cases, the measured quality may be the size or shape of a biological component. In some cases, the system may be used to study epigenomics, DNA methylation, or chromatin accessibility in a biological component. The system may be used for other suitable assays, experiments, and processes.

[0068] In certain embodiments, the system may be used for single-cell analysis on an indirect cell-cell interaction level. For example, an effect of one or more molecules produced from a first cell on a second cell can be analyzed using the system as provided herein. In various embodiments, the system may be used for analyzing direct cell-cell interactions. For example, two or more cells (e.g., a first cell and a second cell) can be in physical contact and the effect or effects of the first cell on the second cell, or vice versa, can be analyzed using the system as disclosed herein. In some embodiments, the system may be used for drug response analysis in a biological component. In certain embodiments, the system may be used for analyzing a biological component’s response to various physiological conditions (e.g., various media, temperature, mechanical stimuli, etc.). In some embodiments the analyte is selected from a plurality of analytes in the fluidic device prior to (a).

[0069] In certain embodiments, one or more polymer precursors may be added to or included with the biological sample. One or more biological samples and one or more polymer precursors may be introduced into the system (e.g., into the fluidic device of the system). The one or more biological samples and the one or more polymer precursors may be introduced into the fluidic device in any order (e.g., in parallel, sequentially, etc.). For example, the biological sample(s) may be introduced prior to the polymer precursor(s), the polymer precursor(s) may be introduced prior to the biological sample(s), the biological sample(s) and polymer precursor(s) may be introduced simultaneously (or substantially simultaneously), or in any other suitable manner or order. In some embodiments, a polymer precursor may include one or more hydrogel precursors, a porogen, and a photoinitiator.

[0070] As used herein, the term “photoinitiator” can denote a species that generates a radical upon photoexcitation. In some cases, a photoinitiator included in a polymer precursor formulation is atype I photoinitiator, that is, a molecule that generates radicals through intramolecular cleavage (e.g., homolysis) upon photoexcitation. In some cases, a photoinitiator included in a polymer precursor formulation is a type II photoinitiator, that is, a molecule that abstract an electron or hydrogen atom from a co-initiator following photoexcitation. Examples of a photoinitiator includes one of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), Irgacure 2959, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO) nanoparticles, 2,2’-azobis[2-methyl-N- (2-hydroxyethyl) promionamide] (VA-086), BAPO-Oli, BAPO-Ona, Eosin-Y, Riboflavin, and combination thereof.

[0071] As used herein, the term “porogen” can denote a species that modulates the porosity of a polymer matrix. A porogen can be dispersed with the reactants before the polymerization process of forming the polymer matrix. Porogens typically diffuse out of polymer matrices following polymerization, leaving pores in the regions that they occupied. Porogen size, concentration, hydrophobicity, and hydrophilicity can thus influence pore density and pore size in polymer matrices. Examples of a porogen can be polyethylene glycol (PEG), 5 arm PEG, 4 arm PEG, 3 arm PEG, and combinations thereof.

[0072] The one or more polymer precursors may be stored and / or introduced separately into the system. In some cases, the one or more polymer precursors may be mixed with the one or more biological components prior to introduction into the system. In various cases, the one or more polymer precursors may be mixed with the one or more biological components after introduction into the system.

[0073] The system may comprise a fluidic device. In some embodiments, the fluidic device may include one or more polymer precursors. In other words, one or more polymer precursors may be disposed within at least a portion of the fluidic device (e.g., within at least a portion of a channel of the fluidic device). In some embodiments, the fluidic device may comprise one or more channels or chambers. In some embodiments, the fluidic device may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 1,000, 10,000 channels or chambers, or any number of channels or chambers between any of the two numbers mentioned herein. In some embodiments, the fluidic device comprises more than 10,000 channels or chambers. As described herein, the fluidic device may include one or more channels. The fluidic device may also, or alternatively, include one or more chambers. The terms channel and chamber may be usedinterchangeably in the disclosure herein unless indicated otherwise. For example, a channel or a chamber of the fluidic device may comprise a first surface, a second surface, or more surfaces.

[0074] A channel or chamber of a fluidic device (also sometimes referred to as a “flow chamber,” “flow channel,” or “reaction chamber,” as opposed to a chamber that is formed from polymer matrix walls within a channel) may receive or be configured to receive a biological sample. FIG. 5 shows a simplified schematic illustration of a surface of a fluidic device (from left to right): a top view 504 of a portion of eight flow channels that span the width (15 fields) where one flow channel 506 is schematically illustrated as an elongated hexagonal boundary that represents a wall of the flow channel a top view of a single field 508, and an exploded top view of a portion of a field 510 with 80 pm diameter dots and a 130 pm hexagonal pitch. Each field is represented by 507 and 508. Each field contains 1536 spots 509. A biological cell 512 is enclosed (i.e. caged) in hydrogel chamber 511.

[0075] FIG. 15 shows a schematic cross-sectional side view illustration of a portion of a channel 300 that may be disposed in at least a portion of a fluidic device of a system as provided herein. The fluidic device may comprise a channel 300. The channel 300 may comprise a first surface 301. Further, the channel 300 may comprise a second surface 302. In some embodiments, the first surface 301 and the second surface 302 are disposed, placed, or positioned opposite of one another (e.g., as depicted in FIG. 15). In some embodiments, a middle spacer layer of double sided adhesive with a cut-out portion can be used to position the first surface 301 and second surface 302 in a facing relationship to at least partly form the flow channel. In some embodiments, the first surface and second surface are substantially parallel, so that the perpendicular distance between them is substantially the same throughout the channel, for example, where chambers are formed. In some embodiments, the perpendicular distance between a first surface and a second surface depends in part and the nature and size of the biological components to be analyzed. In some embodiments, such as, those adapted to analyzing mammalian cells, the perpendicular distance between a first surface and a second surface may be in the range of from 10 pm to 500 pm, or in the range of from 50 pm to 250 pm. In some embodiments, the perpendicular distance between a first surface and a second surface may be in the range of from twice the average size of the biological component to be analyzed to five times the average size of the biological component to be analyzed. In some embodiments, the perpendicular distance between a first surface and a second surface may be in the range of from twice the average size of the largest biologicalcomponent in the biological sample to five times the average size of the largest biological component in the biological sample. In some embodiments, the first surface 301 may be a lower surface. In certain embodiments, the second surface 302 may be an upper surface. The channel 300 may receive a biological sample comprising one or more biological components 50, 51. The channel 300 may receive one or more polymer precursors. As illustrated in FIG. 15, the biological components 50, 51 may include cells. However, as discussed herein, the biological components may include tissues, proteins, nucleic acids, etc. In some embodiments, the first surface 301, the second surface 302, or both surfaces may couple or receive, or be configured to couple or receive, at least one of the one or more biological components 50, 51. In some cases, the first surface 301 may couple or receive, or be configured to couple or receive, a biological component (e.g., biological components 50, 51). In certain cases, the second surface, 302 may couple or receive, or be configured to couple or receive, a biological component (e.g., biological components 50, 51).

[0076] With continued reference to FIG. 15, in some cases, the first surface 301, the second surface 302, or the first surface 301 and the second surface 302 may be functionalized with an adherent substrate. In particular cases, a bottom surface-with respect to gravity (e.g., 301 as shown in FIG. 3)-of the fluidic device includes an adherent substrate. As used herein, an “adherent substrate” can be a substrate that promotes cell adherence. A cell (50, 51) may have a higher affinity for an adherent substrate than for a fluidic device surface (301, 302) on which the adherent substrate is disposed. Many cells readily adhere to constituents of extracellular matrices (ECMs) such as glycosaminoglycans and fibronectin. An adherent substrate can include an ECM biomolecule or a material that includes an ECM biomolecule. In some cases, the cell adherent support is selected from actinin, collagen, fibrinogen, fibronectin, gelatin, ICAM-1, ICAM-2, laminin, osteopontin, paxillin, poly-l-lysine (PLL), poly-d-lysine (PDL), poly-l-ornithine, talin, VCAM-1, vinculin, vitronectin, a cell adherent peptide, or a combination thereof. As a further example, the cell adherent peptide can include a sequence recognized by one or more integrins, for RGD or LRE. An adherent cell can be inputted into a fluidic device and then the cell can settle onto the adherent substrate (601C) of the bottom surface of the fluidic device. After a period of time, the adherent cell can transition to an adherent state where the adherent cell couples to the adherent substrate. In many instances, the transition to the adherent state can be characterized by a change in shape from a generally spherical shape to a non-spherical shape. For example, an adherent cell in the adherent state can be elongated and may include one or more protrusions.

[0077] In certain cases, a channel may have a cross-sectional area that is rectangular, circular, semi-circular, oval cross-section, or other suitably shaped cross-section. In some cases, a channel may have a triangular, square, rectangular, polygonal, or other cross-section. Accordingly, the channel may have three or more internal surfaces. One or more of the internal surfaces may be couple or receive, or be configured to couple or receive, the one or more biological components.

[0078] In some cases, the first surface 301, the second surface 302, or both surfaces 301, 302 may be functionalized, for example, with a coating (e.g., a surface coating). In some embodiments, the surface coating may be a surface polymer. Some non-limiting examples of surface coatings may include a capture reagent (e.g., pyridinecarboxaldehyde (PCA)), a functional group to capture one or more moieties (e.g., a chemical moiety), an acrylamide, an agarose, a biotin, a streptavidin, a strep-tag II, a linker, a functional group comprising an aldehyde, a phosphate, a silicate, an ester, an acid, an amide, an alkyne, an azide, an aldehyde dithiolane, or a combination thereof. In various embodiments, the surface coating may include a functional group to capture one or more moieties. For example, the acrylamide, the agarose, etc. may include such a functional group. In certain embodiments, the surface polymer may comprise polyethylene glycol (PEG), a thiol, an alkene, an alkyne, an azide, or combinations thereof. In various embodiments, the surface polymer may comprise a silane polymer. In some embodiments, the surface polymer may be functionalized with at least one of an oligonucleotide, an antibody, a cytokine, a chemokine, a protein, an antibody derivative, an antibody fragment, a carbohydrate, a toxin, or an aptamer.

[0079] In some cases, the first surface 301, the second surface 302, or both surfaces 301, 302 may comprise one or more barcodes (e.g., nucleic acid barcodes). In some embodiments, the first surface 301, the second surface 302, or both surfaces 301, 302 may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 1,000, 10,000, 50,000, 100,000, 250,000, 500,000, 1,000,000, 2,000,000, 5,000,000, 10,000,000, 15,000,000 barcodes, or any number of barcodes between any of the two numbers mentioned herein. The barcodes may cover an area of about 500 nm2to about 500 pm2. In some embodiments, the first surface 301, the second surface 302, or both surfaces 301, 302 may comprise at most about 10,000,000 total number of barcodes. The barcodes may be different from one another (e.g., each barcode may be unique). In certain embodiments, a first portion or subset of the barcodes may be different from a second portion or subset of the barcodes. There may be 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 1,000, 10,000 portions or subsets of the barcodes, or any number of portions or subsets of the barcodes betweenany of the two numbers mentioned herein. In some cases, a barcode (or a portion / subset of barcodes) may be associated with the location of the barcode on a surface (location coordinates (e.g., x-, y-coordinates) on a surface of a channel). A barcode may be attached to or coupled to the captured biological component. In some embodiments, the barcode may be a unique identifier that distinguishes a biological component from other biological components (e.g., that identifies a first biological component versus a second biological component). In some embodiments, a barcode may comprise a nucleic acid sequence (e.g., common sequence) to capture a biological component, or used in amplification. In some embodiments, a barcode may comprise a unique identifier comprising a unique nucleic acid sequence (e.g., DNA sequence, RNA sequence, etc.), protein tag, antibody, or an aptamer. In some embodiments the barcode may comprise a fluorescent molecule. In some embodiments, a location of the captured biological component may be associated with the unique identifier to, for example, retain spatial information of a biological component.

[0080] In some embodiments, the fluidic device may be a flow cell. For example, the fluidic device may be used for sequencing (e.g., DNA or RNA sequencing). In some embodiments, the fluidic device may be a microfluidic device. In certain embodiments, the fluidic device may be a nanofluidic device.

[0081] The system disclosed herein may comprise one or more energy sources. The energy source may be in communication with the fluidic device. In some embodiments, the energy source may be in optical communication with the fluidic device. In some cases, the energy source can be used to form one or more polymer matrices in the fluidic device (e.g., on or adjacent to a surface of a channel or chamber of the fluidic device). In some embodiments, the energy source may comprise a light generating device, a heat generating device, an electrochemical reaction generating device, an electrode, or a microwave device. A polymer matrix may be formed in a channel of the fluidic device. The energy source may direct or transfer energy to a predetermined position in the fluidic device. The energy may cause or activate the one or more polymer precursors to form a polymer matrix (e.g., to polymerize) in the predetermined position.

[0082] In some embodiments, the polymer matrix may comprise a hydrogel. In some embodiments, the hydrogel may be porous enough, or have pores of a suitable size, to allow movement or transfer of a reagent (e.g., an enzyme, a chemical compound, a small molecule, an antibody, etc.) through the polymer matrix, while the hydrogel may not allow movement or transfer of the biological component (e.g., DNA, RNA, a protein, a cell, etc.) through the polymer matrix.In some embodiments, the pores may have a diameter from 5 nm to 100 nm. In some embodiments, the pores may have a diameter from 5 nm to 10 nm, 10 nm to 20 nm, 20 nm to 30 nm, 30 nm to 40 nm, 50 nm to 60 nm, 60 nm to 70 nm, 70 nm to 80 nm, 80 nm to 90 nm, 90 nm to 100 nm. In some embodiments, the pores may have a diameter larger than 100 nm. In some embodiments, the pores may have a diameter smaller than 5 nm. The reagent may comprise an enzyme or a primer having a size of less than 50 base pairs (bp). A primer may comprise a single-stranded DNA (ssDNA). In some embodiments, a primer may have a size from 5 bp to 50 bp. In some embodiments, a primer may have a size from 5 bp to 10 bp, 10 bp to 20 bp, from 20 bp to 30 bp, 30 bp to 40 bp, or 40 bp to 50 bp. In some embodiments, a primer may have a size of more than 50 bp. In certain cases, a primer may have a size of less than 5 bp. A reagent may comprise a lysozyme, a proteinase K, hexamers (e.g., random hexamers), a polymerase, a transposase, a ligase, a catalyzing enzyme, a deoxyribonuclease, a deoxyribonuclease inhibitor, a ribonuclease, a ribonuclease inhibitor, DNA oligos, deoxynucleotide triphosphates, buffers, detergents, salts, divalent cations, or any other suitable reagent.

[0083] FIG. 16 shows a portion of a system as provided herein including an energy source 203. The embodiment of FIG. 16 may include components that resemble components of FIG. 15 in some respects. For example, the embodiment of FIG. 16 includes a channel 200 that may resemble the channel 300 of FIG. 15. With continued reference to FIG. 16, the channel 200 of the system may include a first surface 201 and a second surface 202. In some embodiments, the energy source 203 may comprise one or more energy emitting portions (e.g., an energy emitting portion 205). In some embodiments, the energy source 203 may comprise an energy emitting surface 210 that includes one or more non-emitting portions (e.g., a non-emitting portion 204). The nonemitting portion 204 may not emit, or be configured to emit, energy. In some embodiments, the emitting portion 205 can emit energy in the form of electromagnetic waves (e.g., microwaves, light, heat, etc.) to at least a portion of the fluidic device. In certain embodiments, the emitting portion 205 can emit energy to the fluidic device. In some embodiments, the fluidic channel may be coupled to on a movable stage. In other embodiments, light may be projected to or onto at least a portion of the fluidic channel to generate one or more polymer matrices. The light may be directed to various parts of the fluidic channel. In some embodiments, the emitting portion 205 may be coupled to an objective (e.g., a microscope objective or lens), where the objective may be moved to different portions of the fluidic device. The objective may provide a shape (e.g., virtualmask) to allow light to form a pattern on the fluidic device, in order to form a polymer matrix similar or complementary to the pattern. In various embodiments, the one or more polymer precursors in the fluidic device or mixed with the biological sample can absorb emitted energy 206. In some embodiments, the emitted energy 206 can form, or be sufficient to form, a polymer matrix from the one or more polymer precursors. For example, a portion of the one or more polymer precursors within the channel 200 of the fluidic device may be activated by the emitted energy and a polymerization reaction may be initiated to form a polymer matrix.

[0084] The energy source (e.g., light source) may be coupled to the fluidic device via an objective (e.g., a microscope objective or lens). The energy source may be directed to a portion of the fluidic channel (e.g., via a movable objective). In some cases, the light source, the objective, and / or the fluidic channel are movable to allow emission of energy to the fluidic channel so as to generate a pattern on at least a portion of a surface of the fluidic device. The polymer matrix may be formed similarly or complementary to the pattern of energy emission.

[0085] In some embodiments, a first polymer matrix 208 can be formed on or adjacent to a biological component 50. In certain embodiments, the first polymer matrix 208 can form a cylindrical analysis chamber or compartment 220 that separates (e.g., physically separates) the biological component 50 from other biological components (e.g., biological components 51, 52, or 53) in the fluidic device. Stated another way, the polymer matrix may compartmentalize the channel (e.g., channel 200) in cooperation with the first surface 201 and the second surface 202. In various embodiments, the polymer matrix may partially surround a biological component. For example, a polymer structure surrounding a biological component may form a closed structure (e.g., a hollow cylinder-shaped polymeric structure) or a partially open structure (e.g., a crescentshaped polymeric structure). In some embodiments, two or more polymer matrices may be formed adjacent to a biological component forming a compartment separating the biological component from other biological components. In certain embodiments, the polymer matrix may comprise or form a wall (e.g., a polymer matrix wall).

[0086] With continued reference to FIG. 16, in some cases, the energy source 203 can, or be configured to, form or produce one or more emitting portions 205 and one or more non-emitting portions 204. The systems disclosed herein may further include a spatial energy modulating element to direct energy from the energy source to one or more targeted portions of the fluidic device. For example, the spatial energy modulating element may be configured to selectively directthe energy from the energy source to form a polymer matrix in a discrete area of the fluidic device. In some embodiments, the discrete area is chosen based on the location of a biological component. In some embodiments, the area of the discrete area is less than the area of the fluidic device. In some embodiments, a biological component is captured within the discrete area. In some embodiments, the size and shape of the discrete area is adjustable according to the size, shape, or other properties of the biological component. In some embodiments, an algorithm is used to determine the shape and size of the discrete area. In some embodiments, the algorithm is a supervised, a self-supervised, or an unsupervised learning algorithm. The spatial energy modulating element may be configured to selectively direct the energy by, for example, inhibiting or preventing energy from being directed to one or more portions other than the one or more targeted portions of the fluidic device. In some embodiments, the spatial energy modulating element may comprise a physical mask. In some cases, the spatial energy modulating element may comprise a virtual mask. In some cases, the spatial energy modulating element may be a spatial light modulator (SLM). In some embodiments, the SLM is a digital micromirror device (DMD). In some embodiments, the SLM is a laser beam steered using a galvanometer. In some embodiments, the SLM is liquid-crystal based.

[0087] In some embodiments, the first surface 201 or the second surface 202 may comprise a detector that detects, or is configured to detect, one or more locations of one or more biological components in the fluidic device (e.g., in the channel 200). In certain embodiments, the energy source 203 can comprise, be coupled to, or be in communication with a detector that detects, or is configured to detect, a location of a biological component in the fluidic device. In some embodiments, the detector may be a microscope objective for imaging the fluidic device. In various embodiments, a mask may be generated using an image obtained from at least a portion of the fluidic device. The mask may allow or permit the energy source 203 to emitting energy in or toward one or more locations or positions where one or more biological components are present on or adjacent the first surface 201. The mask may inhibit or prevent the energy source 203 from emitting energy in or toward one or more locations or positions where one or more biological components are present on or adjacent the first surface 201. In some embodiments, the image may be obtained from a camera (e.g., a digital camera, fluorescent imaging camera, etc.). In some embodiments, the imaging is bright-field imaging, phase-contrast imaging, or fluorescence imaging, or any combination thereof. In some embodiments, the camera may be coupled to,connected to, or in communication with the energy source 203. For example, the camera (not shown) may be in electrical communication with the energy source 203. In some embodiments, the energy source 203 may comprise the camera. In various embodiments, the energy source 203 may comprise a microscope (e.g., a fluorescence microscope, a confocal microscope, lens-free imaging system, a transmission electron microscopy (TEM), a scanning electron microscope (SEM), etc.). The microscope may be used to detect one or more positions of one or more biological components (e.g., in combination with the detector).

[0088] In some embodiments, an algorithm is used to determine where a biological component or analyte is located based on the imaging. In some embodiments, the algorithm is a supervised, a self-supervised, or an unsupervised learning algorithm. In some embodiments, the objective is coupled to an energy source to emit energy to the predetermined portion in the fluidic channel.CRISPR Screening

[0089] “CRISPR” (Clustered Regularly Interspaced Short Palindromic Repeats), first discovered in bacteria as a part of their immune system, is a tool for genetic engineering and allows for precise editing of the DNA in living cells, including insertion, deletion, or replacement of genes. The primary components of the CRISPR system include the Cas (CRISPR-associated) protein and a guide RNA. The most well-known variant is CRISPR-Cas9, where Cas9 acts as the "scissors" to cut DNA at a specific location guided by the RNA. This break prompts the cell's repair machinery to fix the cut, during which alterations can be made to the DNA sequence. Numerous variants have been developed with different properties and applications: 1) CRISPR- Casl2a (formerly Cpfl): this variant has different PAM sequence requirements and generates staggered cuts, unlike the blunt ends of Cas9. It can also process its own CRISPR array without the need for tracrRNA. 2) CRISPR-Casl3 (formerly C2c2): Instead of targeting DNA, this system targets RNA, allowing for post-transcriptional gene silencing. 3) Base Editing: This is a modified version of CRISPR-Cas9, where a base editor protein is fused to Cas9, enabling direct conversion of one DNA base into another without double-strand breaks. 4) Prime Editing: This advanced variant can make precise insertions, deletions, and all possible base-to-base changes, but with lesser off-target effects compared to previous systems. 5) CRISPRa and CRISPRi: These are used for gene activation (CRISPRa) or repression (CRISPRi) by fusing a transcriptional activator or repressor to a nuclease-null Cas protein, altering gene expression without modifying the sequence.6) dCas9-SAM: Synergistic Activation Mediator (SAM) is a fusion of transcriptional activators to dCas9, enhancing its gene activation function.

[0090] “Guide RNAs” are a component of the CRISPR gene-editing system, directing the Cas proteins to their precise DNA targets. Various guide RNA configurations exist. One common configuration is the single guide RNA (sgRNA), which is a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). In some cases, configuration simplifies the system and facilitates multiplexing, where multiple sgRNAs can be used to target multiple genes at once. Other variants include the dual guide RNA system, which employs two separate RNAs: the crRNA to identify the target sequence and the tracrRNA to bind to Cas9. In some cases, there is also a scaffolded guide RNA, which includes additional sequences to the sgRNA to allow for the binding of accessory proteins to modulate gene expression. In addition, there's a cyclically-permutated guide RNA, which is a novel configuration designed to improve editing efficiency in Cas9 systems.

[0091] As used herein, the terms “guide RNA,” “gRNA,” “sgRNA,” and “single guide RNA” can refer to ribonucleotide molecules that include a gRNA sequence. It is worth noting that some gRNA sequences do not activate Cas nucleases for target binding or cleavage when they contain 5’ or 3’ extensions-that is, when they are coupled to additional sequences such as a promoter (e.g., the complement of the second promoter 2103’ in FIG. 21) or mRNA (e.g., mRNA 2102’ in FIG. 21). However, as used herein, the terms “guide RNA” and “gRNA” encompass (1) ribonucleotides that activate Cas nucleases for targeted substrate binding and cleavage; and (2) ribonucleotides that include a gRNA sequence coupled to one or more additional sequences, wherein the gRNA sequence is capable of activating a Cas nuclease for targeted substrate binding and cleavage if provided as a standalone sequence (i.e., as a ribonucleotide consisting solely of the gRNA sequence).

[0092] As used herein, a “gRNA barcode” may be a gRNA sequence contained within a larger molecule. For example, in FIG. 21, 2104’ may be referred to as a gRNA barcode or a gRNA sequence. Alternatively, a gRNA barcode may be a barcode sequence that corresponds to a particular gRNA sequence.

[0093] Provided herein is a method for analyzing a biological component, the method comprising (a) introducing the biological component into a fluidic device. In some embodiments, the method further comprises (b) encapsulating the biological component in a hydrogel chamber.The encapsulating may include synthesizing the hydrogel chamber from a polymer precursor. For example, the method may include introducing a polymer precursor into the fluidic device, and projecting light into the fluidic device with a spatial energy modulating element such that the projected light causes cross-linking of one or more polymer precursors to at least partially form walls of the hydrogel chamber.

[0094] In some embodiments, the biological component comprises (i) a guide RNA barcode associated with a genetic modification of the biological component and (ii) messenger RNA (mRNA). FIG. 21 shows a DNA molecule with coding strand 2100A and template strand 2100B. The DNA molecule (2100A, 2100B) can encode two guide RNA molecules 2110 and 2120. In FIG. 21, the designations ‘D’ and ‘R’ at the end of labels indicate that an element contains DNA or RNA, respectively. The coding strand 2100A of the DNA molecule includes, in order, a first promoter sequence 210 ID, a first coding sequence 2102D, a second promoter sequence 2103D, a second coding sequence 2104D, and a polyadenylation signal 2105D. The template strand 2100B may include a complement of the first promoter sequence 210 ID’, a complement of the first coding sequence 2102D’, a complement of the second promoter sequence 2103D’, a complement of the second coding sequence 2104D’, and a complement of the polyadenylation signal 2105D’. Two transcripts may be generated from the DNA molecule: a first guide RNA molecule 2110 under control of the first promoter 210 ID and a second guide RNA molecule 2120 under control of the second promoter 2103D. The first promoter sequence 2101D and the second promoter sequence 2103D may be promoters for different polymerases. For example the first promoter may be a promoter for polymerase II and the second promoter may be a promoter for polymerase III. Accordingly, transcription initiated at the first promoter sequence 2101D may terminate at a different location than transcription initiated at the second promoter sequence 2103D. The first transcript 2110 may include, in order, an RNA copy of the first coding sequence 2102R, an RNA copy of the second promoter 2103R, an RNA copy of the second coding sequence 2104R, and a capturable sequence 2106R. The RNA copy of the first coding sequence 2102R may be an exogenous mRNA sequence. As used herein, “exogenous mRNA” may also be referred to as an exogenous RNA sequence or a coding portion of an exogenous RNA sequence that is not natively expressed by a cell that contains the DNA molecules 2100A and 2100B. In some embodiments, the exogenous mRNA may consist of a coding portion of an mRNA sequence. The RNA copy of the second coding sequence 2104R may be a gRNA sequence. The gRNA sequence (2104R) mayinclude a crRNA sequence, a linker, and a tracrRNA sequence. As non-limiting examples, the polyadenylation signal 2105D may be an SV40 sequence and the capturable sequence 2106R may be a polyA sequence. The polyadenylation signal 2105D may prompt a cell to generate or append the capturable sequence 2106R onto the first gRNA molecule 2110. The second transcript 2120 may include or consist of a copy of the second coding sequence 2104R.

[0095] It is worth noting that the sequences of the ‘Guide-Seq Elution Product’ from FIG. 2 may correspond to portions of the first DNA molecule 2100A and B of FIG. 21 in the following manner: FBP1 from FIG. 2 may be the complement of the first coding sequence 2102D’ from FIG. 21; U6, U6-1, and U6-2 from FIG. 2 may collectively correspond to the complement of the second promoter sequence 2103D’ from FIG. 21; and sgRNA and tracr from FIG. 2 may collectively correspond to the complement of the second coding sequence 2104D’ from FIG. 21. That is, when a capture probe couples to the capturable RNA sequence 2106R of the first guide RNA molecule 2110 and is extended using at least a portion of the first guide RNA molecule 2110 as a template, the extended capture probe may include a complement of the first coding sequence 2102D’, a complement of the second promoter 2103’, and a complement of the second coding sequence 2104D’. The extended capture probe may be amplified using one or more primers that are targeted to the complement of the second promoter 2103D’ or portions of the complement of the second promoter 2103D’ (corresponding to U6, U6-1, and U6-2 in FIG. 2).

[0096] A further example of a biological component is provided in FIG. 22. This figure depicts a guide RNA molecule 2200 that consists of a tracrRNA sequence 2201, a linker sequence 2202, and a crRNA sequence 2203. The guide RNA molecule 2200 in FIG. 22 may correspond to the second guide RNA molecule 2120 in FIG. 21. In this example, the guide RNA molecule 2200 consists of a guide RNA sequence (i.e., 2201, 2202, and 2203 collectively form a gRNA sequence). The guide RNA molecule 2200 is not fused to an additional sequence such as an exogenous mRNA sequence or a promoter sequence. A portion of the tracrRNA sequence 2201 is hybridized to a capture sequence 2212 of a capture probe 2210. The capture probe 2210 may be a single stranded nucleic acid. The capture probe 2210 may include one or more additional sequences 2211 such as a spatial barcode, a primer binding site, a nuclease recognition sequence, and the like. The capture probe 2210 can be coupled to a surface 2220, such as the top or bottom surface of a fluidic device. The capture probe 2210 can be extended using at least a portion of the gRNA molecule 2200 as a template. The extended capture probe can then be detached from the surface 2220 for amplificationand / or sequencing of the extended capture probe or a derivative of the extended capture probe or a derivative of a complement to the extended capture probe.

[0097] In some embodiments, the method further comprises (c) releasing the guide RNA molecules and mRNA molecules from the biological component. In some embodiments, the fluidic device comprises one or more capture probes. In some embodiments, subsequent to (c), the one or more capture probes capture the guide RNA molecules and the mRNA molecules released from the biological component. More specifically, the capture probes may include capture sequences that are configured to hybridize to guide RNA molecules (e.g., a transcript that includes a guide RNA sequence and optionally further includes an mRNA sequence), mRNA molecules (distinct transcripts from the guide RNA molecules), or to the guide RNA molecules and the mRNA molecules. A chamber may co-enclose a cell with capture probes within a fluidic device. The chamber may include polymer matrix walls that prevent or inhibit diffusion of the guide RNA molecules and mRNA molecules released from the cell. The guide RNA molecules and the mRNA molecules may thus be blocked from diffusing out of a chamber, and may hybridize only to capture probes that are co-enclosed inside of the chamber in which the guide RNA and mRNA were released. A capture probe may be extended using at least a portion of a captured guide RNA molecule or a captured mRNA molecule as a template. Similarly, guide RNA molecules and mRNA molecules may be extended using at least a portion of a capture probe as a template. Extension may include reverse transcription. Reverse transcription reagents may comprise conventional reagents for reverse transcription; namely, a reverse transcriptase (such as, a Moloney murine leukemia virus (MMLV)), dNTPs, optional RNase inhibitor, buffer. Extension may include the use of a DNA polymerase such as Taq polymerase or Pfu polymerase. Extended capture probes, extended guide RNA molecules, and / or extended mRNA molecules may be eluted from a fluidic device and sequenced. In some embodiments, extended capture probes, extended guide RNA molecules, and / or extended mRNA molecules can include a uracil group proximal to the 5’ end so that uracil DNA glycosylase (USER) enzyme can be used for cleavage from the fluidic device. Examples of sequencing methods consistent with the present disclosure include nanopore sequencing, pyrosequencing, sequencing-by-hybridization, sequencing-by-ligation, sequencing-by-synthesis, single-molecule sequencing, digital gene expression, next generation sequencing, shotgun sequencing, Sanger sequencing, ion torrent sequencing, as well as other next- generation-sequencing methods known in the art.

[0098] Capture probes may contain spatial barcode sequences that are uniquely associated with a location within a fluidic device, and which may therefore be associated with a particular cell and / or chamber within the fluidic device. Accordingly, extended capture probes (which contain the spatial barcodes) and RNA extended using the nucleic acid barcodes as templates (which contain complements of the spatial barcodes) may be associated with a particular cell or a chamber. Capture probes may also contain unique molecular identifiers to facilitate mRNA quantitation by normalizing sequencing counts. For example, during sequencing, the number of instances of each guide RNA sequence may be determined based on the number of unique molecular identifier sequences associated with that guide RNA sequence. Alternatively, a method may compare raw sequencing counts of target nucleic acid sequences.

[0099] In some embodiments, the one or more capture probes each comprise a spatial location tag corresponding to a unique location of the capture probe on the fluidic device. In some embodiments, the fluidic device comprises one or more arrays located on a surface of the fluidic device. In some embodiments, each array comprises one or more reaction sites.

[0100] A reaction site can include the spatial location tag. In some cases, the spatial location tag comprises: (a) a first oligonucleotide sequence unique to a spatial location of the reaction site within the array; and (b) a second oligonucleotide unique to the array of the one or more arrays. In some embodiments, the unique location comprises at least a portion of the reaction site. In some embodiments, a capture probe of the one or more capture probes comprises an anti-polyA probe configured to couple with the guide RNA. In some embodiments the method further comprises subsequent to (c), generating a cDNA guide barcode based on the guide RNA barcode and generating a cDNA segment based on the mRNA and then sequencing the cDNA guide barcode and the cDNA segment.

[0101] In some embodiments, (a) comprises introducing a plurality of biological components into the fluidic device. The plurality of biological components may include the biological component. In some embodiments the method further comprises prior to (b) performing one or more assays on the plurality of biological components. In some embodiments, the biological component is selected to at least be partially encapsulated in the hydrogel chamber from the plurality of biological components based on a result of the one or more assays. For example, multiple biological components with varying properties may be introduced into a fluidic device. A user can perform one or more assays on the entire population of biological components. Basedon the results of the one or more assays, specific biological components out of the population may be selected for further analysis. The biological components that are not selected may be washed from the fluidic device.

[0102] In some embodiments the one or more assays comprises a fluorescence assay or a secretion assay. In some embodiments, the secretion assay comprises measuring one or more secretions of the one or more biological components. In some embodiments, the one or more secretions comprise a cytokine (e.g. Interleukin 1 (IL-1), Interleukin 2 (IL-2), Interleukin 3 (IL-3), Interleukin 4 (IL-4), Interleukin 5 (IL-5), Interleukin 6 (IL-6), Interleukin 7 (IL-7), Interleukin 8 (IL-8), Interleukin 9 (IL-9), Interleukin 10 (IL-10), Interleukin 12 (IL-12), Interleukin 13 (IL-13), Interleukin 14 (IL-14), Interleukin 15 (IL-15), Interleukin 16 (IL-16), Interleukin 17A-F (IL-17A- F), Interleukin 17C (IL-17C), Tumor Necrosis Factor alpha (TNF-alpha), Tumor Necrosis Factor beta (TNF-beta), Interferon alpha (IFN-alpha), Interferon beta (IFN-beta), Interferon gamma (IFN- gamma), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Granulocyte Colony- Stimulating Factor (G-CSF), Macrophage Colony-Stimulating Factor (M-CSF), Transforming Growth Factor beta (TGF-beta), and Leukemia Inhibitory Factor (LIF)). In some embodiments, beads coated with antibodies specific to cytokines can be incorporated into hydrogel containers for measuring cytokines secreted by cells. Once the beads bind the cytokines, a sandwich ELISA assay can be performed for measuring the amount of cytokines. Beads coated with antibodies specific to cytokines can be purchased from Biolegend.

[0103] Cell secretion analysis can include disposing a capture surface (e.g., a bead) comprising an affinity reagent (e.g., an aptamer or an antibody) adjacent to a cell, wherein a secretion from the cell couples to the affinity reagent, and detecting the secretion coupled to the affinity reagent. Disposing the capture surface adjacent to the cell can include enclosing or at least partially enclosing the capture surface with the cell within one or more chambers, and optionally removing non-enclosed capture surfaces from the fluidic device that contains the cell. In an exemplary embodiment, the capture surface is a bead. As used herein, the term “bead” can denote a microparticle or a nanoparticle, such as a ceramic, metal, metal oxide, polymer, or saccharide- based 30 to 10000 pm particle. However, further capture surfaces such as nanotubes, nucleic acid nanostructures, and antibody Fc domains may be used. The capture surface affinity reagent can, as non-limiting examples, include antibodies, antibody fragments, aptamers, affimers, or a combination thereof.

[0104] A secretion coupled to a capture surface (e.g., to an affinity reagent of a capture surface) can be detected by contacting the secretion coupled to the capture surface with a labeled ‘detection’ antibody configured to couple to the secretion, and detecting the labeled antibody. Multiple secreted proteins can be detected in a single assay by providing a capture surface or plurality of capture surfaces that include a plurality of affinity reagents configured to bind the plurality of secreted proteins, contacting the plurality of secreted proteins bound to the capture surface or plurality of capture surfaces with a plurality of labeled antibodies configured to bind to the plurality of secreted proteins, and detecting a plurality of labels coupled to the plurality of antibodies.

[0105] Secretion analysis may also be performed with a bispecific binding agent capable of simultaneously binding to a cell and to a secretion. The bispecific binding agent can be coupled to a target cell of interest and then used to capture secretions from the cell. In this way, the bispecific binding agent may couple a secretion to the surface of the cell. The secretion may then be detected, for example by coupling a detectable binding agent such as a fluorescent antibody to the secretion coupled to the surface of the cell, and measuring the detectable binding agent.

[0106] In some embodiments, the biological component comprises a T cell. In some embodiments, the one or more assays comprise measuring one or more surface markers of the one or more biological components. In some embodiments, the one or more surface markers comprise CD3, CD4, CD8, CD19, CD25, CD45, or CD56, or any combination thereof. In some embodiments, the one or more surface markers comprise CD69, CD127, CD28, CD45RA, CD45RO, CD62L, CTLA-4, or PD-1, or any combination thereof. In some embodiments, the one or more assays for measuring a surface marker can include exposing the biological component to an antibody coupled to a dye, wherein the antibody is specific to one or more surface markers such as CD3, CD4, CD8, CD 19, CD25, CD45, or CD56, or any combination thereof, wherein the dye is a fluorescent dye such as an Alexa Fluor dye.

[0107] In some embodiments, the method further comprises (d) introducing an additional biological component into a fluidic device. In some embodiments the method further comprises (e) encapsulating the additional biological component in the hydrogel chamber. In some embodiments, the biological component and the additional biological component are both in the hydrogel chamber. In some embodiments, the one or more assays comprise measuring an interaction between the biological component and an additional biological component. In some embodiments, the interaction comprises a killing of the biological component by the additionalbiological component. In some embodiments, the biological component comprises a T cell. In some embodiments, the biological component comprises a CD8+ T cell. In some embodiments, the biological component comprises a CAR-T cell. In some embodiments, the interaction comprises a killing of the additional biological component by the biological component. In some embodiments, the additional biological component comprises a CD8+ T cell. In some embodiments, the additional biological component comprises a CAR-T cell. In some embodiments, the interaction comprises a proliferation of the biological component or the additional biological component. In some embodiments, the interaction comprises a physical contact between the biological component and the additional biological component. In some embodiments, biological components that have been killed can be identified with Sytox dead cell stain that is absorbed by dead cells.

[0108] It is understood that the term “proliferation rate” may include a measure of a lack of proliferation. Proliferation rate can be determined by counting cells that are at least partially enclosed by the one or more chambers generated during an assay. For example, one or more cells can be counted periodically (e.g., with fluorescence or brightfield imaging) following at least partial enclosure within one or more chambers to determine a rate of change in the number of cells. Separate proliferation rates can be determined for each cell or collection of cells enclosed by a unique chamber or collection of chambers. In some embodiments, cells may be stained with a membrane or intracellular dye for determining proliferation by dye dilution so that an independent measure of cell proliferation may be obtained. Exemplary intracellular dyes for dye dilution include, but are not limited to, Hoechst 33342, carboxyfluorescein succinimidyl ester (CFSE), and the like. After counts are recorded for each chamber, further assays may be conducted on the clonal populations within the chambers to identify the cell types, for example, by an assessment of cell surface proteins, cell protein secretions, transcriptome, or the like.

[0109] In some embodiments, the biological component comprises a neuron. In some embodiments the method further comprises measuring Ca2+ signaling of the neuron. In some embodiments, the method further comprises measuring ATP release of the neuron. In some embodiments, the method further comprises screening the neuron for deoxyribonucleic acid (DNA) double strand breaks. In some embodiments, the method further comprises determining a morphological classification of the neuron. In some embodiments, the morphological classification comprises a unipolar neuron, a bipolar neuron, a multipolar neuron, or a pseudo-unipolar neuron.

[0110] In some aspects, a method includes measuring activation of one or more cells. Cellular activation can be detected using numerous assays disclosed herein, including surface marker expression, soluble factor secretion, transcriptomic analysis, proliferation or changes in proliferation, changes in morphology, change in cytotoxicity, or a combination thereof. As nonlimiting examples, these methods are broadly amenable to detecting activation caused by an interaction between an analyte and a biological material.

[0111] In some embodiments, the biological component comprises a dendritic cell. In some embodiments, the method further comprises measuring an antigen presentation of the dendritic cell. In some embodiments, the method further comprises measuring activation of a T cell by the dendritic cell.

[0112] In some embodiments, the genetic modification comprises a CRISPR-modification. In some embodiments, the biological component expresses Cas9 or a variant thereof.

[0113] In some embodiments, the method further comprises imaging the biological component. In some embodiments, the method further comprises imaging the fluidic device.

[0114] In some embodiments, the method further comprises correlating a result of the one or more assays with the cDNA guide barcode for the biological component. In some embodiments, the method further comprises degrading the hydrogel chamber when the cytokine signal is below a predetermined threshold.

[0115] Provided herein is a fluidic device for analyzing a biological component, comprising a hydrogel chamber encapsulating the biological component. In some embodiments, the biological component comprises (i) a guide RNA associated with a genetic modification of the biological component and (ii) messenger RNA (mRNA). In some embodiments, the fluidic device comprises one or more capture probes configured to capture the guide RNA and mRNA upon release from the biological component. In some embodiments, the one or more capture probes each comprise a spatial location tag corresponding to a unique location of the capture probe on the fluidic device. In some embodiments, the fluidic device comprises one or more arrays. In some embodiments, an array of the one or more arrays is located at an intersection of a row on a surface of the fluidic device. In some embodiments, the array comprises one or more reaction sites. In some embodiments, a reaction site of the one or more reaction sites comprises the spatial location tag. In some embodiments, the spatial location tag comprises: (a) a first oligonucleotide sequence unique to a spatial location of the reaction site within the array; and (b) a second oligonucleotideunique to the array of the one or more arrays. In some embodiments, the unique location comprises at least a portion of the reaction site. In some embodiments, a capture probe of the one or more capture probes comprises an anti-polyA probe configured to couple with the guide RNA. In some embodiments, the fluidic device further comprises a plurality of biological components. In some embodiments, the plurality of biological components comprises the biological component. In some embodiments, the biological component is selected at least partially based on a result of one or more assays. In some embodiments, the one or more assays comprises a fluorescence assay. In some embodiments, the one or more assays comprise a secretion assay. In some embodiments, the secretion assay comprises measuring one or more secretions of the one or more biological components. In some embodiments, the one or more secretions comprise a cytokine (for example, Absolutely, here is a list of some cytokines written in paragraph form: Interleukin 1 (IL-1), Interleukin 2 (IL-2), Interleukin 3 (IL-3), Interleukin 4 (IL-4), Interleukin 5 (IL-5), Interleukin 6 (IL-6), Interleukin 7 (IL-7), Interleukin 8 (IL-8), Interleukin 9 (IL-9), Interleukin 10 (IL-10), Interleukin 12 (IL-12), Interleukin 13 (IL-13), Interleukin 14 (IL-14), Interleukin 15 (IL-15), Interleukin 16 (IL- 16), Tumor Necrosis Factor alpha (TNF-alpha), Tumor Necrosis Factor beta (TNF-beta), Interferon alpha (IFN-alpha), Interferon beta (IFN-beta), Interferon gamma (IFN- gamma), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Granulocyte Colony- Stimulating Factor (G-CSF), Macrophage Colony-Stimulating Factor (M-CSF), Transforming Growth Factor beta (TGF-beta), and Leukemia Inhibitory Factor (LIF)). In some embodiments, the biological component comprises a T cell. In some embodiments, the one or more assays comprise measuring one or more surface markers of the one or more biological components. In some embodiments, the one or more surface markers comprise CD3, CD4, CD8, CD19, CD25, CD45, or CD56, or any combination thereof. In some embodiments, the one or more surface markers comprise CD28, CD45RO, CD45RA, CD 127, CD62L, CTLA-4, or PD-1, or any combination thereof. In some embodiments, the one or more assays for measuring a surface marker can include exposing the biological component to an antibody coupled to a dye, wherein the antibody is specific to one or more surface markers such as CD3, CD4, CD8, CD19, CD25, CD45, or CD56, or any combination thereof, wherein the dye is fluorescent dye such as an Alexa Fluor dye.

[0116] In some embodiments, the one or more assays comprise measuring an interaction between the biological component and an additional biological component of the one or morebiological components. In some embodiments, the interaction comprises a killing of the biological component by the additional biological component. In some embodiments, the additional biological component comprises a T cell. In some embodiments, the biological component comprises a CD8+ T cell. In some embodiments, the biological component comprises a CAR-T cell. In some embodiments, the interaction comprises a killing of the additional biological component by the biological component. In some embodiments, the additional biological component comprises a CD8+ T cell. In some embodiments, the additional biological component comprises a CAR-T cell. In some embodiments, the interaction comprises a proliferation of the biological component or the additional biological component. In some embodiments, the interaction comprises a physical contact between the biological component and the additional biological component.

[0117] In some embodiments, the biological component comprises a neuron. In some embodiments, the genetic modification impacts Ca2+ signaling of the neuron. In some embodiments, the genetic modification impacts ATP release of the neuron. In some embodiments, the genetic modification impacts deoxyribonucleic acid (DNA) double strand breaks of the neuron. In some embodiments, the neuron comprises a unipolar neuron, a bipolar neuron, a multipolar neuron, or a pseudo-unipolar neuron.

[0118] In some embodiments, the biological component comprises a dendritic cell. In some embodiments, the genetic modification impacts an antigen presentation of the dendritic cell. In some embodiments, the genetic modification impacts activation of a T cell by the dendritic cell.

[0119] In some embodiments, the genetic modification comprises a CRISPR-modification. In some embodiments, the biological component expresses Cas9 or a variant thereof.Degradable hydrogels for functional assays

[0120] Degradable hydrogels can be utilized to facilitate controlled cell interactions, allowing a step-wise approach for evaluating cell-cell interactions, such as the potency of a potential cytotoxic cell over a series of cell populations. This can provide a precise and tunable way to scrutinize the serial killing capacity of different analytes in a biomimetic environment, allowing advancements in the capacity to design and conduct complex biological assays with implications for numerous fields, including biomedical research, pharmacology, and oncology.

[0121] Provided herein are methods for facilitating a controlled interaction of one or more biological components. In some cases, the method comprises (a) inputting an analyte and one or more biological materials into a fluidic device. In some embodiments, the fluidic device comprises a hydrogel chamber. In some embodiments, the hydrogel chamber comprises: (i) at least a portion of the analyte and (ii) at least a portion of a first biological material of the one or more biological materials. In some embodiments, the analyte and the first biological material are physically separated by a first hydrogel polymer wall. In some embodiments, the method further comprises (b) degrading at least a portion of the first hydrogel polymer wall. In some embodiments, the method further comprises (c) detecting an interaction between the analyte and the first biological material.

[0122] In some cases, the method comprises (a) detecting an interaction between the analyte and a first biological material of the one or more biological materials in a fluidic device. In some embodiments, the method further comprises (b) forming a hydrogel polymer wall that physically separates the analyte from a second biological material of the one or more biological materials.

[0123] In some cases, the method comprises (a) providing a fluidic device comprising the analyte and one or more biological materials. In some embodiments the fluidic device comprises a hydrogel chamber. In some embodiments the fluidic device comprises (i) at least a portion of the analyte and (ii) at least a portion of a first biological material of the one or more biological materials. In some embodiments, the method further comprises (b) imaging the hydrogel chamber to determine whether the analyte kills the first biological material.

[0124] In some embodiments, the method comprises (d) inputting a second biological material into the fluidic device. In some embodiments, the analyte and the second biological material are physically separated by a second hydrogel polymer wall. In some embodiments, the method further comprises (e) degrading the hydrogel chamber and (f) forming a second hydrogel chamber around at least a portion of the analyte and at least a portion of the second biological material. In some embodiments, the method further comprises (g) degrading at least a portion of the second hydrogel polymer wall. In some embodiments, the method further comprises (h) detecting an interaction between the analyte and the second biological material. In some embodiments, the method further comprises repeating these steps for additional materials of the one or more biological materials. In some cases, the steps are repeated until the analyte is exhausted. In some cases, an exhausted analyte will no longer kill target cells or biological materials. In some instances, the interaction iskilling and an analyte that has killed one or more target cells can be analyzed for guide RNA and mRNA to correlate a particular genetic modification with an effectiveness in cell killing.

[0125] Optically cleavable hydrogels can be used in serial killing assays. In some cases, optically cleavable hydrogels respond to light wavelengths, allowing controlled degradation. This can enable systematic testing of an agent's cytotoxic impact on distinct cell groups. This approach can be used to measure cellular response and can be used in applications such as cell biology, pharmacology, and oncology.

[0126] In some embodiments, an analyte and a first biological material are surrounded by a hydrogel chamber. Within the hydrogel chamber, the analyte and the first biological material can be physically separated by a first hydrogel polymer wall. In some embodiments, the method further comprises degrading at least a portion of the first polymer wall, thereby resulting in an interaction (or lack thereof) between the analyte and the first biological material. In some cases, a second polymer wall can be formed around the analyte. The analyte and a second biological material can be physically separated by the second polymer wall. In some embodiments, the method further comprises detecting an interaction between the analyte and the second biological material. In some embodiments, the method further comprises repeating the steps for additional materials of the one or more biological materials.

[0127] In some embodiments, the analyte is a cell. In some embodiments, the analyte is an antigen targeting cell. In some embodiments, the analyte is a CD8+ T cell or NK cell. In some embodiments, the analyte is a genetically engineered cell. In some embodiments, the analyte is a CAR-T cell. In some embodiments, the first biological material is a cell. In some embodiments, the first biological material is an antigen presenting cell. In some embodiments, the first biological material is a cancer cell. In some embodiments, the first biological material is an antibody or antigen binding fragment thereof. In some embodiments, the antibody or antibody binding fragment thereof is coupled to a bead.Porosity of hydrogel chambers and polymer walls

[0128] In some embodiments, the hydrogel polymer wall prohibits the interaction between the analyte and the biological material. In some embodiments, hydrogel porosity is selected to permit passage of selected reagents while at the same time preventing the passage of other reagents or objects, such as, a cell. In some embodiments, hydrogel porosity is selected to prevent the passageof biological cells but to permit the passage of reagents, including proteins, such as polymerases. In some embodiments, such reagents permeable to a polymer matrix wall comprise lysozyme, proteinase K, random hexamers, polymerases, transposases, ligases, deoxynucleotide triphosphates, buffers, cell culture media, or divalent cations. In some embodiments, the at least one polymer matrix comprises pores that are sized to allow diffusion of a reagent through the at least one polymer matrix but are too small to allow DNA or RNA for analysis to traverse the pores (having a size of greater than 100 nucleotides or base pairs, or greater than 300 nucleotides or base pairs). In some embodiments, crosslinking the polymer chains of the hydrogel structure forms a hydrogel matrix having pores (i.e., a porous hydrogel matrix). In some versions, the size of the pores in the hydrogel structures may be regulated or tuned and may be formulated to encapsulate sufficiently large genetic material, such as cells or nucleic acids (e.g., of greater than about 300 base pairs), but to allow smaller materials, such as reagents, or smaller sized nucleic acids (e.g., of less than about 50 base pairs), such as primers, to pass through the pores, thereby passing in and out of the hydrogel structures. In some embodiments, the hydrogels can have any pore size having a diameter sufficient to allow diffusion of the above-listed reagents through the structure while retaining the nucleic acid molecules greater than 500 nucleotides or base pairs in length. In some embodiments, the hydrogel structure can be swollen when the hydrogel is hydrated. The sizes of the pores can then change depending on the water content in the hydrogel of the hydrogel structure. In some embodiments, the pores have a diameter of from about 10 nm to about 100 nm. In some embodiments, the pore size of the hydrogel structures is tuned by varying the ratio of the concentrations of polymer precursors to the concentration of crosslinkers, varying pH, salt concentrations, temperature, light intensity, and the like, by routine experimentation. In some embodiments, the average diameter of pores of a polymer matrix wall prevent passage of molecules having a molecular weight of 25 kiloDaltons (kDa) or greater; or having a molecular weight of 50 kDa or greater; or having a molecular weight of 75 kDa or greater; or having a molecular weight of 100 kDa or greater; or having a molecular weight of 150 kDa or greater.

[0129] In some embodiments, the analyte, the first biological material, or both, is coupled to the hydrogel polymer wall. In some embodiments, the fluidic device comprises a flow cell. In some embodiments the method further comprises obtaining one or more genetic materials from the analyte. In some embodiments, the method further comprises amplifying the one or more genetic materials. In some embodiments, the amplifying occurs in the fluidic device. In someembodiments, the method further comprises sequencing the one or more genetic materials. In some embodiments, the sequencing occurs in the fluidic device. In some embodiments, the genetic material comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In some embodiments, the RNA comprises messenger RNA (mRNA) or microRNA (miRNA). In some embodiments, the interaction comprises a killing, activation, suppression or binding of the biological material by the analyte. In some embodiments, the interaction comprises a killing, activation, suppression or binding of the first biological material by the analyte. In some embodiments, the interaction comprises a killing, activation, suppression or binding of the second biological material by the analyte.Hydrogel Compositions

[0130] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises an optically cleavable hydrogel. In some embodiments, the degrading comprises exposing the hydrogel polymer wall to UV light. In some embodiments, the hydrogel chamber and the hydrogel polymer wall are made of different materials. In some embodiments, the degrading in (b) does not degrade the hydrogel chamber. In some embodiments, the method further comprises degrading the hydrogel chamber. In some embodiments, the method further comprises imaging the analyte, the first biological material, the hydrogel chamber, the fluidic device, or any combination thereof. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a cPEG monomer. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (I):(I).

[0131] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (II):(II).

[0132] In some embodiments, n is between about 0 to about 100, or optionally n is between about 5 to about 50. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (III):(III).

[0133] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (IV):

[0134] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (V):

[0135] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (VI):

[0136] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (VII):

[0137] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (VIII):

[0138] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (IX):

[0139] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (X):(X).

[0140] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (XI):

[0141] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (XII):(XII).

[0142] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (XIII):(XIII).

[0143] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer, the monomer comprising: an oligomeric domain comprising three or more arms, wherein each arm of said oligomeric domain comprises a degradable unit and a crosslinkable unit, wherein the crosslinkable unit of an arm of the three or more arms is configured to crosslink with another crosslinkable unit of another polymer precursor in response to a first stimulus, thereby obtaining the polymerized form of the monomer, and wherein the degradable unit is configured to be cleaved in response to a second stimulus, thereby solubilizing the polymerized form of the monomer. In some embodiments, the oligomeric domain comprises four or more arms. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a degradable functional group. In some embodiments, said degradable function groupcomprises disulfide, Beta-thioether ester, Amidomethylol and vicinal diol, Vicinal diol, Alginate backbone, Dextran backbone, Chitosan backbone, Hyaluronic acid backbone, Chondroitin sulfate backbone, or Carboxy methyl cellulose backbone, or a combination thereof. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a hydrogel macromonomer. In some embodiments, the hydrogel macromonomer comprises cPEG, cSEL- BTEEC, cSEL-DHEBA, cSEL-diol, cSEL-alginate, cSEL-dextran, cSEL-chitosan, cSEL- hyaluronic acid, cSEL-chondroitin sulfate, or cSEL-cellulose, or a combination thereof. In some embodiments, the degradation unit is degraded by inputting a degradation reagent into the fluidic device. In some embodiments, the degradation reagent comprises DTT, TCEP, BME, GSH, DMEM, RPMI, PBS buffer, DMEM, RPMI, PBS buffer, sodium (meta)periodate, Alginate lyase (enzyme), Dextranase, Lysozyme and chitinase, Hyaluronidase, Chondroitinase, or Cellulases, or a combination thereof. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises at least one beta-thioether ester. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a PEG-macromonomer containing beta-thioether esters. In some embodiments, the beta-thioether ester is formed by reacting an acrylate with a thiol. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a Michael donor. In some embodiments, the Michael donor is PEG-thiol. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a cSEL beta-thioether ester with one beta-thioether ester per arm. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises are formed from any material that comprises a PEG with a Michael acceptor chain. In some embodiments, the Michael acceptor chain comprises PEG-acrylamide, PEG-vinyl sulfone, PEG- maleimide, or PEG -carbonyl acrylic, or any combination thereof. In some embodiments, the hydrogel chamber or the hydrogel polymer wall is degradable by cleavage of disulfide bonds. In some embodiments, the disulfide bonds are cleavable by one or more reducing agents. In some embodiments, the one or more reducing agents comprise DTT, TCEP, BME, or GSH, or any combination thereof. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises one or more arms each comprising one or more amides. In some embodiments, the hydrogel chamber or the hydrogel polymer wall is degradable by oxidative cleavage of vicinal diol by sodium (meta)periodate. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a photocleavable 4-arm PEG-macromonomer. In some embodiments, the hydrogel chamber or the hydrogel polymer wall is photodegradable via an ortho-nitrobenzyl moiety. Insome embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a Coumarin- based photodegradable macromonomer. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a 4-arm PEG-acrylamide comprising one or more disulfides. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises one or more cage disulfide bonds in a hydrogel cage formation. In some embodiment, the hydrogel cages degrade using light and a photoinitiator. In some embodiments, the hydrogel chamber or the hydrogel polymer wall enables hydrogel formation. In some embodiments, the hydrogel enables spatiotemporal control of hydrogel cage degradation, therefore enabling selective retention of cells with a single hydrogel formulation. In some embodiments, upon exposure to light, photogenerated radicals’ initial multiple fragmentation and disulfide exchange reactions, thereby permitting and promoting photodeformation, pho to welding and photodegradation of the hydrogel chamber or the hydrogel polymer wall. In some embodiments, one or more polymer precursors enable formation of the hydrogel chamber or the hydrogel polymer wall. In some embodiments, the hydrogel exhibits a chemical or physical change in response to an external stimulus. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a photolabile nitrobenxyl ester which lyses upon photon absorption, thereby allowing a user to exogenously control degradation of the hydrogel chamber or the hydrogel polymer wall. In some embodiments, the method further comprises controlling a network degradation of the hydrogel chamber or the hydrogel polymer wall by concentration of a photoinitaitor infused into the hydrogel chamber or the hydrogel polymer wall. In some embodiments, the first hydrogel polymer wall comprises a shape configured to contain the first biological material. In some embodiments, the fluidic device comprises a top layer, a bottom layer, and a spacer layer. In some embodiments, the spacer layer includes a cutout region, where the spacer layer is sandwiched in between the bottom layer and the top layer to form a channel in the cut-out region. In some embodiments, the hydrogel chamber is at least partly formed by the top layer and the bottom layer. In some embodiments, the hydrogel chamber and the hydrogel polymer wall are the same material. In some embodiments, the hydrogel chamber and the hydrogel polymer wall are different materials.

[0144] In some embodiments, the hydrogel chamber is made of a first material that degrades upon exposure to a first stimulus. In some cases, a hydrogel polymer wall is made of a second material that degrades upon exposure to a second stimulus. The first stimulus and second stimulus can be different. In some cases, the first stimulus comprises light, and the second stimuluscomprises a degradation reagent. In some cases, the first stimulus comprises a degradation reagent, and the second stimulus comprises light. In some cases, the first stimulus comprises a first degradation reagent, and the second stimulus comprises a second degradation reagent different from the first degradation reagent. In some cases, the first stimulus comprises light in a first wavelength range, and the second stimulus comprises a light in a second wavelength range different from the first wavelength range.Computer Systems

[0145] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 17 shows a computer system 1501 that may be programmed or otherwise configured to perform methods described herein. The computer system 1501 can regulate various aspects of the present disclosure, such as, for example, identifying a biological component, detecting a barcode, generating a spatial modulating element (e.g., a mask), providing energy from an energy source, or detecting or measuring a local parameter using a sensor. The detector may be a camera (e.g., a fluorescent camera), such as a charged coupled device (CCD) camera capable of collecting optical signals and position information from a plurality of sources distributed over a planar region. The computer system 1501 can be an electronic device of a user or a computer system that may be remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0146] The computer system 1501 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 1505, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 1501 also includes memory or memory location 1510 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 1515 (e.g., hard disk), communication interface 1520 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1525, such as cache, other memory, data storage and / or electronic display adapters. The memory 1510, storage unit 1515, interface 1520 and peripheral devices 1525 are in communication with the CPU 1505 through a communication bus (solid lines), such as a motherboard. The storage unit 1515 can be a data storage unit (or data repository) for storing data. The computer system 1501 can be operatively coupled to a computer network (“network”) 1530 with the aid of the communication interface 1520. The network 1530 can be the Internet, an internet and / or extranet, or an intranet and / or extranet that may be in communication with the Internet. The network 1530 in some cases may bea telecommunication and / or data network. The network 1530 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 1530, in some cases with the aid of the computer system 1501, can implement a peer-to-peer network, which may enable devices coupled to the computer system 1501 to behave as a client or a server.

[0147] The CPU 1505 can execute a sequence of machine -readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 1510. The instructions can be directed to the CPU 1505, which can subsequently program or otherwise configure the CPU 1505 to implement methods of the present disclosure. Examples of operations performed by the CPU 1505 can include fetch, decode, execute, and writeback.

[0148] The CPU 1505 can be part of a circuit, such as an integrated circuit. One or more other components of the system 1501 can be included in the circuit. In some cases, the circuit may be an application specific integrated circuit (ASIC).

[0149] The storage unit 1515 can store files, such as drivers, libraries, and saved programs. The storage unit 1515 can store user data, e.g., user preferences and user programs. The computer system 1501 in some cases can include one or more additional data storage units that are external to the computer system 1501, such as located on a remote server that may be in communication with the computer system 1501 through an intranet or the Internet.

[0150] The computer system 1501 can communicate with one or more remote computer systems through the network 1530. For instance, the computer system 1501 can communicate with a remote computer system of a user (e.g., a laptop, a personal computer, a tablet, or a mobile phone). Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 1501 via the network 1530.

[0151] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 1501, such as, for example, on the memory 1510 or electronic storage unit 1515. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 1505. In some cases, the code can be retrieved from the storage unit 1515 and stored on the memory 1510 for ready access by the processor 1505. In some situations,the electronic storage unit 1515 can be precluded, and machine-executable instructions are stored on memory 1510.

[0152] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a precompiled or as-compiled fashion.

[0153] Aspects of the systems and methods provided herein, such as the computer system 1501, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and / or associated data that may be carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0154] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, suchas main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier- wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0155] The computer system 1501 can include or be in communication with an electronic display 1535 that comprises a user interface (UI) 1540 for providing, for example, an image of a biological component, a barcode, a signal or measurement of a local parameter. Examples of UI’s include, without limitation, a graphical user interface (GUI) and web-based user interface.

[0156] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 1505. The algorithm can, for example, identify a biological component, detect a barcode, generate a spatial modulating element (e.g., a mask), provide energy from an energy source, detect or measure a local parameter using a sensor, etc.EXAMPLES

[0157] The following illustrative examples are representative of embodiments of the devices and methods described herein and are not meant to be limiting in any way.Example 1. CROP-Seq in Hydrogel Chambers

[0158] The systems and methods described herein can achieve single-cell resolution for CRISPR screening by combining 1) transcriptome profiling, and 2) guide-RNA sequencing of individual cells at scale (FIG. 1A). A mix of cells (101, 102, and 103) with different guide RNAs (106, 107, and 108) can be disposed within a portion of a channel of the fluidic device. In thisfigure, cell 101 expresses guide RNA 107, cell 102 expresses guide RNA 108, and cell 103 expresses guide RNA 106. The fluidic device can comprise one or more polymer precursors along with an array of spatial capture probes on a surface of the fluidic device. An energy source in communication with the fluidic device can direct or transfer energy to the fluidic device, thereby causing the polymer precursors to polymerize where the energy is projected. In some cases, the polymerization results in one or more gel chambers (111, 112, and 113). The gel chambers can encapsulate one or more cells where each chamber encapsulates at least a portion of a reaction zone that includes a single type of spatial capture probe.

[0159] Caged cells can be lysed and mRNAs (105) and guide RNAs (106) from the cell can be hybridized to one or more capture probes located on a surface of the fluidic device. The mRNAs (105) may be referred to as endogenous mRNAs or endogenous mRNA molecules. Different guide RNAs (106, 107, 108) are present in each three gel chambers (111, 112, 113). The mRNAs (105) and guide RNAs (106) can be analyzed (120). Analysis can include sequencing. The sequencing can be performed on the fluidic device. Alternatively, samples can be eluted off of the flow cell and then sequenced. In some cases, each capture probe comprises a spatial tag associated with a location of the capture probe on the flow cell. The spatial tag can capture either guide RNAs or mRNA captured at the same spot, allowing for sequencing based guide identification corresponding with whole transcriptome analysis of an individual cell.Example 2. CROP-Seq and Functional Readout in Hydrogel Chambers

[0160] The systems and methods described herein can achieve single-cell resolution for CRISPR screening by combining 1) transcriptome profiling, 2) guide-RNA sequencing, and 3) functional readouts of individual cells at scale (FIG. IB). A mix of cells (101, 102, and 103) with different guide RNAs (106, 107, and 108) can be disposed within a portion of a channel of the fluidic device. In this figure, cell 101 expresses guide RNA 107, cell 102 expresses guide RNA 108, and cell 103 expresses guide RNA 106. The fluidic device can comprise one or more polymer precursors. An energy source in communication with the fluidic device can direct or transfer energy to the fluidic device, thereby causing the polymer precursors to polymerize where the energy is projected. In some cases, the polymerization results in one or more gel chambers (111, 112, and 113). The gel chambers can encapsulate one or more cells.

[0161] In some cases, one or more assays (such as a fluorescence or secretion assay) can be performed on the cells. A subpopulation of the cells can be selected based on results of the assays.The subpopulation of cells can be caged and subsequently analyzed. The gel chambers can be formed prior to or subsequent to the one or more assays. The gel chambers can be formed only around the subpopulation of cells. In some cases, cages are degraded depending on results of the one or more assays, leaving only the selected subpopulation of cells in the cages.

[0162] Caged cells can be lysed and mRNAs (105) and guide RNAs (106, 107, 108) from the cell can be hybridized to one or more capture probes located on a surface of the fluidic device. Different guide RNAs (106, 107, 108) are present in each three gel chambers (111, 112, 113). The mRNAs (105) and guide RNAs (106) can be analyzed (120). Analysis can include sequencing. The sequencing can be performed on the fluidic device. Alternatively, samples can be eluted off of the flow cell and then sequenced. In some cases, each capture probe comprises a spatial tag associated with a location of the capture probe on the flow cell. The spatial tag can be shared between guide RNAs and mRNA captured on the same spot, allowing for sequencing based guide identification corresponding with whole transcriptome analysis of an individual cell.Example 3. Library Preparation for Guide-Seq and mRNA-seq

[0163] One purpose of this embodiment is to prepare from one sample, a sequencing library for the guide RNAs and the mRNA (FIG. 2). Starting with the elution product from Example 1 or 2, the elution product is amplified with TSO and U6-1 primers (5: 1). In some embodiments, the TSO primer can be referred to as whole transcriptome primer and the U6-1 primer can be referred to as a gene specific primer. 1 p L is reserved for Guide seq and the rest is utilized for standard mRNA library preparation. For the guide-seq sample, nested PCR is performed with the U6-2 primer. 10 ng of the PCR product is PCR’d with i5-P5 and i7-P7 primers to prepare the library. PCR is followed by double cut SPRI bead purification. The mRNA library is sequenced to about 100 million reads and the guide library is sequenced to about 2 million reads.Example 4. CROP-Seq in Hydrogel Chambers of guide library transduced Jurkats

[0164] One purpose of this embodiment is to achieve single-cell resolution for CRISPR screening by combining 1) transcriptome profiling, and 2) guide-RNA sequencing of individual Jurkat cells at scale. Following the protocols described in Examples 2 & 3, library preparation products were generated from Jurkats cells transduced with non-targeting guide RNA, targeting guide RNA or nothing. FIG. 3 illustrates the concentration and size distribution of the guide RNA and mRNA libraries. In some embodiments, the fluidic device can undergo 2 elution events to elute two batches of elution material that can be pooled together for subsequent analysis orprocessing. FIG. 4 shows the results of sequencing the mRNA library. A dramatic difference in the total UMI per cell count between muti-cell, no-cage, no-cell, single cell, and undetected samples was observed. Further, the average UMI per gene / barcode in the caged single cells was significantly higher than the no cage sample. FIG. 6 illustrates fluorescent based discrimination of non-transduced, targeting guide, or non-targeting guide transduced jurkats. Both targeting guide and non-targeting guide cells are GFP positive while non-transduced cells have no fluorescence. Targeted guide cells stained positive with CD45 alexafluor red About 43.7% of cells were transduced guide cells based on their expression of GFP and green fluorescence (FIG. 7). Transduced jurkat cells in hydrogel chambers were determined by the jurkat’s expression of green fluorescence (see left hand side of FIG. 8). Based on the green population, the jurkat’s with targeted and non-targeted guide RNAs were determined by their expression of red fluorescence (see right hand side of FIG. 8). Jurkats transduced with targeting guide RNA made up 41% of the cell in hydrogel chambers. Further both non-targeting and targeting guide UMI’s were primarily detected inside of cages (FIG. 9). Guide UMI mapping matched image-based cell identification in cages (FIG. 10). In support of the assay’s specificity, no correlation was found between cell size and UMI count (FIG. 11). A strong correlation was observed with hydrogel chamber / barcode overlap and UMI count (FIG. 12). To further enrich guide-seq specificity non-target specific (FIG. 13), or non-target specific (FIG. 14) guide RNA containing jurkats, a threshold of 13% overlap or higher between the hydrogel chamber and the barcode was applied demonstrating a significant refinement in enriched UMI’s.Example 5. gRNA Analysis of Three Closely Related Cell Lines

[0165] This example covers gRNA analysis on two HeLa (adherent cervical cancer cell) lines. The first HeLa cell line was transfected with a vector encoding red fluorescent protein and a first guide RNA sequence (‘RFP cell line’). The second HeLa cell line was transfected with a vector encoding green fluorescent protein and a second guide RNA sequence (‘GFP cell line’). A subset of cells in the first and second HeLa cell lines were not successfully transfected, and therefore did not express a fluorescent protein or guide RNA. The first and second guide RNA sequences included polyA terminal sequences to facilitate capture.

[0166] The HeLa cells were loaded into four channels of an eight channel fluidic device. The first and fourth channels were loaded with a 50:50 mixture of the RFP and GFP cell lines. The second channel was loaded with the RFP cell line. The third channel was loaded with the GFP cellline. The bottom surface of each channel included a fibronectin coating to promote HeLa cell adherence to the channel. The top surface of each channel included arrays of capture probes that each contained (i) polyT sequences for gRNA and mRNA capture and (ii) spatial location tags associated with locations of the capture probes on the surface of the fluidic device. Within each channel, the cells were enclosed within cylindrical hydrogel chambers by photopolymerizing a polymer precursor in circular patterns surrounding the cells. Each chamber enclosed a single cell. The chamber included a porous polymer wall where the pores were sufficiently small to retain the cell in the chamber and allowed lower molecular weight reagents to pass through such as buffers, media, and lysing reagents. The channels were subjected to multiple buffer and media wash steps to remove cells that were not enclosed by hydrogel chambers. The cells were then incubated for twelve hours to allow the HeLa cells to adhere to the fibronectin coatings on the bottom surfaces of the channels. The cells were subjected to brightfield imaging at lOx magnification. The cells were also subjected to fluorescence imaging to measure GFP and RFP expression.

[0167] Then, the cells were lysed. gRNA and mRNA released from the cells was captured on capture probes co-enclosed in the hydrogel chambers with the cells. The capture probes were then extended using the gRNA and mRNA as templates, cleaved from the surfaces of the channels, and eluted from the channels for amplification and sequencing. Each sequence was associated with a particular chamber and cell based on its spatial barcode sequence. Sequence reads were returned as raw counts of amplified gRNA sequences.

[0168] FIG. 18 is a scatter plot of RFP and GFP fluorescence signals from chamber-enclosed cells in the first fluidic device channel. Images of exemplary chambers with RFP+ and GFP+ cells are shown at the top of the figure. The images include manually-drawn cell borders to enhance the contrast between the cells and fluidic device background. The x- and y-axes of the plot are log2 intensities of RFP fluorescence signal and GFP fluorescence signal, respectively, from individual cells. The cells were classified into four categories based on their fluorescence profiles. Cells with RFP fluorescence intensities of at least 4 fluorescence units and GFP fluorescence intensities of less than 22 5fluorescence units were classified as RFP cells (portion of plot labeled 1801). Cells with GFP fluorescence intensities of at least 225fluorescence units and RFP fluorescence intensities of less than 4 fluorescence units were classified as GFP cells (portion of plot labeled 1802). Cells with GFP fluorescence intensities of less than 22 5fluorescence units and RFP fluorescence intensities of less than 4 fluorescence units were classified as ‘blank’ cells (portionof plot labeled 1803), that is, cells that did not include the first vector or the second vector, and which therefore did not express RFP, GFP, the first gRNA, or the second gRNA. Cells with GFP fluorescence intensities greater than 22 5fluorescence units and RFP fluorescence intensities greater than 4 fluorescence units were excluded from analysis (portion of plot labeled 1804).

[0169] Total sequencing counts (not normalized) for the first and second guide RNAs are shown in FIGS. 19A-D. In each of these figures, the x-axis provides LOGio counts of first or second gRNA reads and the y-axis provides the number of chambers. FIG. 19A is a plot of total sequencing reads of the first gRNA per chamber in the second channel. FIG. 19B is a plot of total sequencing reads of the second gRNA per chamber in the third channel. As can be seen from these plots, gRNA sequencing reads per chamber in the second and third channels were monomodal, with the majority of chambers having 103and 105gRNA sequencing reads. FIGS. 19C and 19D are plots of total sequencing reads of the first guide RNA and second guide RNA, respectively, per chamber in the first channel.

[0170] FIG. 20 is a plot of gRNA sequencing reads from individual cells in the first channel. Within this figure, the x-axis provides the difference in LOGio values of sequencing counts of the first guide RNA and LOGio sequencing counts of the second guide RNA, and the y-axis provides normalized counts of chambers. In FIG. 20, separate traces are provided for RFP cells, GFP cells, and blank cells. Cells were classified according to their RFP and GFP fluorescence intensities as shown in FIG. 18.. The RFP cell population (which expressed the first gRNA), the GFP cell population (which expressed the second gRNA), and the blank cell population (which did not express gRNA) exhibit distinct peaks within FIG. 20, demonstrating that the presently disclosed methods can distinguish between cell populations that express different guide RNA molecules.Exemplary Embodiments

[0171] The embodiments listed below are exemplary embodiments of the systems and methods described herein, and do not limit the description above:

[0172] Embodiment 1. A method for analyzing a biological component, the method comprising: (a) introducing the biological component into a fluidic device; (b) in the fluidic device, encapsulating the biological component in a hydrogel chamber, wherein the biological component comprises a guide ribonucleic acid (RNA) associated with a genetic modification of the biological component; and (c) releasing the guide RNA from the biological component.

[0173] Embodiment 2. The method of embodiment 1, wherein the fluidic device comprises one or more capture probes.

[0174] Embodiment 3. The method of embodiment 2, wherein subsequent to (c), the one or more capture probes capture the guide RNA released from the biological component.

[0175] Embodiment 4. The method of embodiment 2, wherein the one or more capture probes each comprise a spatial location tag corresponding to a unique location of the capture probe on the fluidic device.

[0176] Embodiment 5. The method of embodiment 4, wherein the fluidic device comprises one or more arrays located on a surface of the fluidic device, wherein each array comprises one or more reaction sites, and wherein a reaction site of the one or more reaction sites comprises the spatial location tag, and wherein the spatial location tag comprises: (a) a first oligonucleotide sequence unique to a spatial location of the reaction site within the array; and (b) a second oligonucleotide unique to the array of the one or more arrays.

[0177] Embodiment 6. The method of embodiment 5, wherein the unique location comprises at least a portion of the reaction site.

[0178] Embodiment 7. The method of embodiment 2, wherein a capture probe of the one or more capture probes comprises an anti-polyA probe configured to couple with the guide RNA.

[0179] Embodiment 8. The method of embodiment 1, wherein the guide RNA comprises a single guide RNA (sgRNA).

[0180] Embodiment 9. The method of embodiment 1, wherein the guide RNA comprises an exogenous messenger RNA (mRNA), and wherein the exogenous mRNA comprises a single guide RNA (sgRNA) sequence.

[0181] Embodiment 10. The method of embodiment 1, wherein the guide RNA comprises an exogenous messenger RNA (mRNA), and wherein the exogenous mRNA comprises a barcode encoding a single guide RNA (sgRNA) sequence.

[0182] Embodiment 11. The method of embodiment 1, wherein the biological component further comprises an endogenous messenger RNA (mRNA), and wherein the endogenous mRNA is released from the biological component in (c).

[0183] Embodiment 12. The method of embodiment 11, further comprising subsequent to (c), generating a cDNA guide barcode based on the guide RNA and generating a cDNA segment based on the mRNA and then sequencing the cDNA guide barcode and the cDNA segment.

[0184] Embodiment 13. The method of embodiment 12, further comprising: eluting the cDNA guide barcode and the cDNA segment from the fluidic device to form an elution mixture; and amplifying the cDNA guide barcode and the cDNA segment to form an amplification mixture using a whole transcriptome primer configured to initiate amplification with the cDNA segment and using a gene specific primer configured to initiate amplification with the cDNA guide barcode.

[0185] Embodiment 14. The method of embodiment 12, further comprising correlating a result of one or more assays performed on the biological component with the cDNA guide barcode of the biological component.

[0186] Embodiment 15. The method of embodiment 1, further comprising introducing additional biological components into the fluidic device, and wherein the biological component is at least partially selected from the additional biological components based on a result of one or more assays performed on the biological component and the additional biological components.

[0187] Embodiment 16. The method of embodiment 15, wherein the performing of the one or more assays occurs prior to (b).

[0188] Embodiment 17. The method of embodiment 15, wherein the performing of the one or more assays occurs subsequent to (b).

[0189] Embodiment 18. The method of embodiment 17, further comprising, based on the result of the one or more assays, (i) degrading the hydrogel chamber encapsulating the biological component or (ii) degrading an additional chamber encapsulating an additional biological component.

[0190] Embodiment 19. The method of embodiment 15, wherein the one or more assays comprises a fluorescence assay.

[0191] Embodiment 20. The method of embodiment 15, wherein the one or more assays comprise a secretion assay.

[0192] Embodiment 21. The method of embodiment 20, wherein the secretion assay comprises measuring one or more secretions of the biological component and the additional biological components.

[0193] Embodiment 22. The method of embodiment 21, wherein the one or more secretions comprise a cytokine.

[0194] Embodiment 23. The method of embodiment 22, further comprising degrading the hydrogel chamber encapsulating the biological component when a signal associated with the cytokine is below a threshold value.

[0195] Embodiment 24. The method of embodiment 22, further comprising, subsequent to (b), removing additional biological components having cytokine signals below a threshold value.

[0196] Embodiment 25. The method of embodiment 20, wherein the biological component comprises a T cell.

[0197] Embodiment 26. The method of embodiment 15, wherein the one or more assays comprise measuring one or more surface markers of the one or more biological components.

[0198] Embodiment 27. The method of embodiment 26, wherein the one or more surface markers comprise CD3, CD4, CD8, CD19, CD25, CD45, or CD56, or any combination thereof.

[0199] Embodiment 28. The method of embodiment 15, wherein the one or more assays comprise measuring an interaction between the biological component and an additional biological component.

[0200] Embodiment 29. The method of embodiment 28, wherein the additional biological component is encapsulated in the hydrogel chamber with the biological component.

[0201] Embodiment 30. The method of embodiment 28, wherein the interaction comprises a killing of the biological component by the additional biological component.

[0202] Embodiment 31. The method of embodiment 30, wherein the biological component comprises a T cell.

[0203] Embodiment 32. The method of embodiment 31, wherein the biological component comprises a CD8+ T cell.

[0204] Embodiment 33. The method of embodiment 31, wherein the biological component comprises a CAR-T cell.

[0205] Embodiment 34. The method of embodiment 28, wherein the interaction comprises a killing of the additional biological component by the biological component.

[0206] Embodiment 35. The method of embodiment 34, wherein the additional biological component comprises a CD8+ T cell.

[0207] Embodiment 36. The method of embodiment 34, wherein the additional biological component comprises a CAR-T cell.

[0208] Embodiment 37. The method of embodiment 28, wherein the interaction comprises a proliferation of the biological component or the additional biological component.

[0209] Embodiment 38. The method of embodiment 28, wherein the interaction comprises a physical contact between the biological component and the additional biological component.

[0210] Embodiment 39. The method of embodiment 1, wherein the biological component comprises a neuron.

[0211] Embodiment 40. The method of embodiment 39, further comprising measuring Ca2+ signaling of the neuron.

[0212] Embodiment 41. The method of embodiment 39, further comprising measuring ATP release of the neuron.

[0213] Embodiment 42. The method of embodiment 39, further comprising screening the neuron for deoxyribonucleic acid (DNA) double strand breaks.

[0214] Embodiment 43. The method of embodiment 39, further comprising determining a morphological classification of the neuron.

[0215] Embodiment 44. The method of embodiment 43, wherein the morphological classification comprises a unipolar neuron.

[0216] Embodiment 45. The method of embodiment 43, wherein the morphological classification comprises a bipolar neuron.

[0217] Embodiment 46. The method of embodiment 43, wherein the morphological classification comprises a multipolar neuron.

[0218] Embodiment 47. The method of embodiment 43, wherein the morphological classification comprises a pseudo-unipolar neuron.

[0219] Embodiment 48. The method of embodiment 1, wherein the biological component comprises a dendritic cell.

[0220] Embodiment 49. The method of embodiment 48, further comprising measuring an antigen presentation of the dendritic cell.

[0221] Embodiment 50. The method of embodiment 48, further comprising measuring activation of a T cell by the dendritic cell.

[0222] Embodiment 51. The method of embodiment 1, wherein the genetic modification comprises a CRISPR-modification.

[0223] Embodiment 52. The method of embodiment 1, wherein the biological component expresses Cas9 or a variant thereof.

[0224] Embodiment 53. The method of embodiment 1, further comprising imaging the biological component.

[0225] Embodiment 54. The method of embodiment 1, further comprising imaging the fluidic device.

[0226] Embodiment 55. The method of embodiment 1, further comprising introducing one or more polymer precursors into the fluidic device.

[0227] Embodiment 56. The method of embodiment 55, wherein the one or more polymer precursors comprise a crosslinker, a porogen, and a photoinitiator.

[0228] Embodiment 57. The method of embodiment 55 or 56, wherein the one or more polymer precursors are introduced into the fluidic device at a same time as the introducing of the biological component in (a).

[0229] Embodiment 58. A fluidic device for analyzing a biological component, comprising:

[0230] a hydrogel chamber encapsulating the biological component, wherein the biological component comprises a guide ribonucleic acid (RNA) associated with a genetic modification of the biological component; and

[0231] one or more capture probes configured to capture the guide RNA upon release from the biological component.

[0232] Embodiment 59. The fluidic device of embodiment 58, wherein the one or more capture probes each comprise a spatial location tag corresponding to a unique location of the capture probe on the fluidic device.

[0233] Embodiment 60. The fluidic device of embodiment 59, wherein the fluidic device comprises one or more arrays on a surface of the fluidic device, wherein each array comprises one or more reaction sites, and wherein a reaction site of the one or more reaction sites comprises the spatial location tag, and wherein the spatial location tag comprises: (a) a first oligonucleotide sequence unique to a spatial location of the reaction site within the array; and (b) a second oligonucleotide unique to the array of the one or more arrays.

[0234] Embodiment 61. The fluidic device of embodiment 59, wherein the unique location comprises at least a portion of the reaction site.

[0235] Embodiment 62. The fluidic device of embodiment 58, wherein a capture probe of the one or more capture probes comprises an anti-polyA probe configured to couple with the guide RNA.

[0236] Embodiment 63. The fluidic device of embodiment 58, wherein the guide RNA comprises a single guide RNA (sgRNA).

[0237] Embodiment 64. The fluidic device of embodiment 58, wherein the guide RNA comprises an exogenous messenger RNA (mRNA), and wherein the exogenous mRNA comprises a single guide RNA (sgRNA) sequence.

[0238] Embodiment 65. The fluidic device of embodiment 58, wherein the guide RNA comprises an exogenous messenger RNA (mRNA), and wherein the exogenous mRNA comprises a barcode encoding a single guide RNA (sgRNA) sequence.

[0239] Embodiment 66. The fluidic device of embodiment 58, wherein the biological component further comprises a messenger RNA (mRNA), and wherein the one or more capture probes are configured to capture the mRNA upon release from the biological component.

[0240] Embodiment 67. The fluidic device of embodiment 5586, further comprising additional biological components, wherein the biological component is at least partially selected from the additional biological components based on a result of one or more assays performed on the biological component and the additional biological components.

[0241] Embodiment 68. The fluidic device of embodiment 67, wherein the one or more assays comprises a fluorescence assay.

[0242] Embodiment 69. The fluidic device of embodiment 67, wherein the one or more assays comprise a secretion assay.

[0243] Embodiment 70. The fluidic device of embodiment 69, wherein the secretion assay comprises measuring one or more secretions of the biological component and the additional biological components.

[0244] Embodiment 71. The fluidic device of embodiment 70, wherein the one or more secretions comprise a cytokine.

[0245] Embodiment 72. The fluidic device of embodiment 71, wherein the biological component comprises a T cell.

[0246] Embodiment 73. The fluidic device of embodiment 67, wherein the one or more assays comprise measuring one or more surface markers of the one or more biological components.

[0247] Embodiment 74. The fluidic device of embodiment 73, wherein the one or more surface markers comprise CD3, CD4, CD8, CD19, CD25, CD45, or CD56, or any combination thereof.

[0248] Embodiment 75. The fluidic device of embodiment 67, wherein the one or more assays comprise measuring an interaction between the biological component and an additional biological component.

[0249] Embodiment 76. The fluidic device of embodiment 75, wherein the additional biological component is encapsulated in the hydrogel chamber with the biological component.

[0250] Embodiment 77. The fluidic device of embodiment 75, wherein the interaction comprises a killing of the biological component by the additional biological component.

[0251] Embodiment 78. The fluidic device of embodiment 77, wherein the biological component comprises a T cell.

[0252] Embodiment 79. The fluidic device of embodiment 78, wherein the biological component comprises a CD8+ T cell.

[0253] Embodiment 80. The fluidic device of embodiment 78, wherein the biological component comprises a CAR-T cell.

[0254] Embodiment 81. The fluidic device of embodiment 75, wherein the interaction comprises a killing of the additional biological component by the biological component.

[0255] Embodiment 82. The fluidic device of embodiment 81, wherein the additional biological component comprises a CD8+ T cell.

[0256] Embodiment 83. The fluidic device of embodiment 81, wherein the additional biological component comprises a CAR-T cell.

[0257] Embodiment 84. The fluidic device of embodiment 75, wherein the interaction comprises a proliferation of the biological component or the additional biological component.

[0258] Embodiment 85. The fluidic device of embodiment 75, wherein the interaction comprises a physical contact between the biological component and the additional biological component.

[0259] Embodiment 86. The fluidic device of embodiment 58, wherein the biological component comprises a neuron.

[0260] Embodiment 87. The fluidic device of embodiment 86, wherein the genetic modification impacts Ca2+ signaling of the neuron.

[0261] Embodiment 88. The fluidic device of embodiment 86, wherein the genetic modification impacts ATP release of the neuron.

[0262] Embodiment 89. The fluidic device of embodiment 86, wherein the genetic modification impacts deoxyribonucleic acid (DNA) double strand breaks of the neuron.

[0263] Embodiment 90. The fluidic device of embodiment 86, wherein the neuron comprises a unipolar neuron.

[0264] Embodiment 91. The fluidic device of embodiment 86, wherein the neuron comprises a bipolar neuron.

[0265] Embodiment 92. The fluidic device of embodiment 86, wherein the neuron comprises a multipolar neuron.

[0266] Embodiment 93. The fluidic device of embodiment 86, wherein the neuron comprises a pseudo-unipolar neuron.

[0267] Embodiment 94. The fluidic device of embodiment 58, wherein the biological component comprises a dendritic cell.

[0268] Embodiment 95. The fluidic device of embodiment 94, wherein the genetic modification impacts an antigen presentation of the dendritic cell.

[0269] Embodiment 96. The fluidic device of embodiment 94, wherein the genetic modification impacts activation of a T cell by the dendritic cell.

[0270] Embodiment 97. The fluidic device of embodiment 58, wherein the genetic modification comprises a CRISPR-modification.

[0271] Embodiment 98. The fluidic device of embodiment 58, wherein the biological component expresses Cas9 or a variant thereof.

[0272] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. Itshould be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method for analyzing a biological component, the method comprising:(a) introducing the biological component into a fluidic device;(b) in the fluidic device, encapsulating the biological component in a hydrogel chamber, wherein the biological component comprises a guide ribonucleic acid (RNA) molecule associated with a genetic modification of the biological component; and(c) releasing the guide RNA molecule from the biological component.

2. The method of claim 1, wherein the fluidic device comprises one or more capture probes.

3. The method of claim 2, wherein subsequent to (c), the one or more capture probes capture the guide RNA molecule released from the biological component.

4. The method of claim 2, wherein the one or more capture probes each comprise a spatial location tag corresponding to a unique location of the capture probe on the fluidic device.

5. The method of claim 4, wherein the fluidic device comprises one or more arrays located on a surface of the fluidic device, wherein each array comprises one or more reaction sites, and wherein a reaction site of the one or more reaction sites comprises the spatial location tag, and wherein the spatial location tag comprises: (a) a first oligonucleotide sequence unique to a spatial location of the reaction site within the array; and (b) a second oligonucleotide unique to the array of the one or more arrays.

6. The method of claim 5, wherein the unique location comprises at least a portion of the reaction site.

7. The method of claim 2, wherein a capture probe of the one or more capture probes comprises an anti-polyA probe configured to couple with the guide RNA.

8. The method of claim 1, wherein the guide RNA molecule comprises a single guide RNA (sgRNA) sequence.

9. The method of claim 1, wherein the biological component further comprises an endogenous messenger RNA (mRNA) molecule, and wherein the endogenous mRNA is released from the biological component in (c).

10. The method of claim 9, further comprising subsequent to (c), generating a cDNA guide barcode based on the guide RNA molecule and generating a cDNA segment based on theendogenous mRNA molecule and then sequencing the cDNA guide barcode and the cDNA segment.

11. The method of claim 10, further comprising: eluting the cDNA guide barcode and the cDNA segment from the fluidic device to form an elution mixture; and amplifying the cDNA guide barcode and the cDNA segment to form an amplification mixture using a whole transcriptome primer configured to initiate amplification with the cDNA segment and using a gene specific primer configured to initiate amplification with the cDNA guide barcode.

12. The method of claim 10, further comprising correlating a result of one or more assays performed on the biological component with the cDNA guide barcode of the biological component.

13. The method of claim 1, further comprising introducing additional biological components into the fluidic device, and wherein the biological component is at least partially selected from the additional biological components based on a result of one or more assays performed on the biological component and the additional biological components.

14. The method of claim 13, wherein the performing of the one or more assays occurs prior to (b).

15. The method of claim 13, wherein the performing of the one or more assays occurs subsequent to (b).

16. The method of claim 15, further comprising, based on the result of the one or more assays, (i) degrading the hydrogel chamber encapsulating the biological component or (ii) degrading an additional chamber encapsulating an additional biological component.

17. The method of claim 13, wherein the one or more assays comprises a fluorescence assay.

18. The method of claim 13, wherein the one or more assays comprise a secretion assay.

19. The method of claim 18, wherein the secretion assay comprises measuring one or more secretions of the biological component and the additional biological components.

20. The method of claim 19, wherein the one or more secretions comprise a cytokine.

21. The method of claim 20, further comprising degrading the hydrogel chamber encapsulating the biological component when a signal associated with the cytokine is below a threshold value.

22. The method of claim 20, further comprising, subsequent to (b), removing additional biological components having cytokine signals below a threshold value.

23. The method of claim 18, wherein the biological component comprises a T cell.

24. The method of claim 13, wherein the one or more assays comprise measuring one or more surface markers of the one or more biological components.

25. The method of claim 24, wherein the one or more surface markers comprise CD3, CD4, CD8, CD 19, CD25, CD45, or CD56, or any combination thereof.

26. The method of claim 13, wherein the one or more assays comprise measuring an interaction between the biological component and an additional biological component.

27. The method of claim 26, wherein the additional biological component is encapsulated in the hydrogel chamber with the biological component.

28. The method of claim 26, wherein the interaction comprises a killing of the biological component by the additional biological component.

29. The method of claim 28, wherein the biological component comprises a T cell.

30. The method of claim 29, wherein the biological component comprises a CD8+ T cell.

31. The method of claim 29, wherein the biological component comprises a CAR-T cell.

32. The method of claim 26, wherein the interaction comprises a killing of the additional biological component by the biological component.

33. The method of claim 32, wherein the additional biological component comprises a CD8+ T cell.

34. The method of claim 32, wherein the additional biological component comprises a CAR-T cell.

35. The method of claim 26, wherein the interaction comprises a proliferation of the biological component or the additional biological component.

36. The method of claim 26, wherein the interaction comprises a physical contact between the biological component and the additional biological component.

37. The method of claim 1, wherein the biological component comprises a neuron.

38. The method of claim 37, further comprising measuring Ca2+ signaling of the neuron.

39. The method of claim 37, further comprising measuring ATP release of the neuron.

40. The method of claim 37, further comprising screening the neuron for deoxyribonucleic acid (DNA) double strand breaks.

41. The method of claim 37, further comprising determining a morphological classification of the neuron.

42. The method of claim 41 , wherein the morphological classification comprises a unipolar neuron.

43. The method of claim 41, wherein the morphological classification comprises a bipolar neuron.

44. The method of claim 41 , wherein the morphological classification comprises a multipolar neuron.

45. The method of claim 41, wherein the morphological classification comprises a pseudounipolar neuron.

46. The method of claim 1 , wherein the biological component comprises a dendritic cell.

47. The method of claim 46, further comprising measuring an antigen presentation of the dendritic cell.

48. The method of claim 46, further comprising measuring activation of a T cell by the dendritic cell.

49. The method of claim 1, wherein the genetic modification comprises a CRISPR-modification.

50. The method of claim 1 , wherein the biological component expresses Cas9 or a variant thereof.

51. The method of claim 1 , further comprising imaging the biological component.

52. The method of claim 1 , further comprising imaging the fluidic device.

53. The method of claim 1, further comprising introducing one or more polymer precursors into the fluidic device.

54. The method of claim 53, wherein the one or more polymer precursors comprise a crosslinker, a porogen, and a photoinitiator.

55. The method of claim 53, wherein the one or more polymer precursors are introduced into the fluidic device at a same time as the introducing of the biological component in (a).