Patent Application
20210348217
The invention, in some aspects, includes systems, methods and components of molecular recorders that encode the timing of transcriptional activity into the sequence of RNA, which can then enable a sequencing-based readout of the internal dynamics of cells.
Despite several new methods for recording cell-state information into sequences of DNA in living cells, no method has been available that can record the absolute timing of cellular events into nucleic acid form in eukaryotic cells. All molecular recorders to date use DNA as a substrate, and record the occurrence of cellular events into the sequence of the DNA (1-10). These methods have shown promise for positioning events within the cellular lineage: because the substrates are DNA based, newly created DNA molecules retain the edits of the parent molecule, so the locations of events within the lineage can be inferred by sequencing the DNA at an endpoint and reconstructing the phylogeny of the reporter. However, these methods are largely incapable of reporting on the timings of cellular events in absolute time. The only methods of molecular recording that have so far achieved absolute timing are incompatible with eukaryotic cell biology, and have resolutions on the order of days, which is too slow for tracking the dynamics of most cellular processes (5).
According to an aspect of the invention, RNA-based molecular recording systems are provided. The systems include a reporter RNA (repRNA) and a predetermined enzyme, wherein an alteration in an original composition of the repRNA indicates one or more of (a) an age of the repRNA and (b) a response of the repRNA to an applied stimulus. In some embodiments, the repRNA comprises an editing array in the 3′ UTR of a target RNA, wherein (i) the editing array comprises one or more engineered binding sites; and (ii) the editing array comprises one or more selectively favored substrates of the predetermined enzyme. In certain embodiments, the target RNA is an endogenous RNA. In certain embodiments, the target RNA is an mRNA. In some embodiments, the editing array comprises at least one of an adenosine-rich editing array and a cytosine-rich editing array. In some embodiments, the predetermined enzyme is attached to a binding polypeptide capable of binding to the engineered binding site in the repRNA editing array. In some embodiments, the engineered binding site is an engineered MS2 binding site. In certain embodiments, the binding polypeptide is an MS2 Capsid protein (MCP) and is capable of binding the MS2 binding sites engineered into the repRNA editing array. In some embodiments, the editing array comprises an adenosine-rich editing array and the predetermined enzyme comprises an Adenosine Deaminase Acting on RNA (ADAR) enzyme. In certain embodiments, the editing array comprises a cytosine-rich editing array and the predetermined enzyme comprises a Cytosine Deaminase Acting on RNA (CDAR) enzyme. In some embodiments, the ADAR or CDAR enzyme is a modified ADAR2 enzyme or modified CDAR enzyme, respectively. In some embodiments, the modified ADAR enzyme is a modified human ADAR2 enzyme or a modified mouse ADAR2 enzyme. In certain embodiments, the predetermined enzyme is ADAR or CDAR and the molecular recording system comprises an MCP-ADAR fusion protein or an MCP-CDAR fusion protein, respectively. In some embodiments, the predetermined enzyme is ADAR E488QT490A, and the molecular recording system comprises an MCP-ADAR E488QT490A fusion protein. In some embodiments, the alteration in an original composition of the repRNA comprises a sequence edit in the repRNA sequence. In some embodiments, the sequence edit in the repRNA sequence comprises one or more adenosine to inosine conversions in the repRNA sequence. In certain embodiments, the editing array sequence is capable of accumulating sequence edits over time, and wherein determining the number of accumulated sequence edits in the editing array over a time period determines the age of the repRNA. In some embodiments, determining one or more edits in the editing array sequence over a time period indicates a repRNA response to a stimulus. In some embodiments, the number of edits in the editing array sequence corresponds to a temporal record of activation of a promoter that generates the repRNAs, and a number of edits in the repRNA corresponds to the length of time elapsed since the activation of the promoter. In certain embodiments, the number of repRNA edits in a test repRNA compared to the number of repRNA edits in a control repRNA indicates the time since promoter activation of the test repRNA and the time since promoter activation of the control repRNA, wherein a different number of edits in the test repRNA compared to the control repRNA indicates a difference in the time period since the activation by the promoter of the test repRNA compared to the time period since the activation by the promoter of the control repRNA. In some embodiments, the stimulus an electrical stimulus, a chemical stimulus, a biological stimulus, a signaling molecule, a signaling chemical, a temperature stimulus, or a light stimulus. In some embodiments, the stimulus activates a promoter and activation of the promoter generates new repRNAs. In some embodiments, the target RNA encodes a detectable protein. In certain embodiments, the detectable protein comprises a fluorescent protein. In some embodiments, the alteration in the repRNA composition comprises a change in the repRNA sequence, wherein the change comprises one or more of: (i) a modification of one or more nucleic acids in the repRNA RNA sequence, and (ii) one or more of a substitution, deletion, and addition of a nucleic acid in the repRNA RNA sequence. In certain embodiments, the repRNA composition is determined using a sequencing means. In some embodiments, the sequencing means comprises single cell sequencing. In some embodiments, the determined repRNA composition is compared to a control repRNA composition and detection of one or more differences between the repRNA composition and the control repRNA composition indicates a change in the repRNA composition. In certain embodiments, the change in the repRNA comprises one or more adenosine to inosine conversions in the repRNA sequence. In some embodiments, the number of changes in the repRNA composition corresponds to the temporal history of activity of a promoter that generates a population of repRNAs. In some aspects of the invention any embodiment of an aforementioned system is included in a cell. In some embodiments a repRNA is in a cell. In certain embodiments, a predetermined enzyme is also in the cell. In some embodiments, the cell is one or more of: a vertebrate cell, a mammalian cell, and a human cell. In some embodiments, wherein the cell is an excitable cell. In certain embodiments, the cell is one or more of: a neuron, a CNS cell, a PNS cell, a muscle cell, an endocrine cell, an immune system cell, an epidermal cell, a kidney cell, a liver cell, and a cardiac cell. In some embodiments, the cell is an in vitro cell.
According to another aspect of the invention, a cell is provided that includes any embodiment of any aforementioned aspect of an RNA-based molecular recording system. In some embodiments, the cell is one or more of: a vertebrate cell, a mammalian cell, a human cell, an excitable cell, a neuron, a CNS, cell, a PNS cell, a muscle cell, a endocrine cell, an immune system cell, an epidermal cell, a kidney cell, a liver cell, a cardiac cell, and an in vitro cell. In some embodiments, wherein the cell is an in vitro cell.
According to another aspect of the invention, a vector is provided that includes one or both of the repRNA and predetermined enzyme set forth in any embodiment of any aforementioned aspect of an RNA-based molecular recording system.
According to another aspect of the invention, a cell that includes any vector of an aforementioned aspect of the invention. In certain embodiments, the cell is one or more of: a vertebrate cell, a mammalian cell, and a human cell, an excitable cell, a neuron, a CNS, cell, a PNS cell, a muscle cell, an endocrine cell, an immune system cell, an epidermal cell, a kidney cell, a liver cell, a cardiac cell, and an in vitro cell.
According to another aspect of the invention, methods of RNA-based molecular recording in a cell are provided. The methods including one or more of any embodiment of a repRNA and predetermined enzyme of any aspect of an aforementioned RNA-based molecular recorder system, and determining the presence of an alteration in the original composition of the repRNA. In some embodiments, including the repRNA and predetermined enzyme in a cell comprises one of more of: expressing and delivering. In certain embodiments, the editing enzyme is bound to an endogenous RNA. In some embodiments, the editing array is inserted into the 3′-UTR of an endogenous RNA. In some embodiments, the RNA comprises mRNA. In some embodiments, the method also includes determining an age of the RepRNA. In certain embodiments, the method also includes determining a response of the repRNA to an applied stimulus. In some embodiments, a means for the determining comprises detecting one or more alternations in the repRNA composition. In certain embodiments, the alteration in the repRNA composition comprises a change in the repRNA sequence, wherein the change comprises one or more of: (i) a modification of one or more nucleic acids in the repRNA RNA sequence, and (ii) one or more of a substitution, deletion, and addition of a nucleic acid in the repRNA RNA sequence.
Additional aspects of the invention include systems and/or use of RNA as a substrate for molecular recording. In some embodiments systems of the invention are used to determine the age of one or more RNAs by altering the composition of the sequence of the RNA. In certain embodiments, methods of the invention use of a system of the invention to monitor the age of RNA in a cell using RNA editing, which may additionally include using the age of RNAs to infer the transcriptional history of the cell. In certain aspects, the invention includes methods of recording multiple different signals on the same RNA; using stimulus-responsive dimerization domains to record the total amount of a given signal that a cell has observed during the lifetime of a particular RNA; and combining both an age-recording system with a stimulus-responsive dimerization system to make a system that can record the time course the application of arbitrary stimuli into the sequence of the RNA. In certain aspects, the invention includes an RNA-based molecular reporting system in which a predetermined enzyme is attached to an endogenous RNA, and the age of the RNA can then to be determined based on detected modifications to the endogenous nucleotide bases of the RNA sequence. Such an embodiment need not include an engineered editing array and does not require the RNA sequence to be an “engineered” RNA sequence.
Additional aspects of the invention include use of a set of 12 adenosines that were edited with much higher rate constants than other adenosines in the repRNA experiments described herein. These “high-edit” adenosines were found to be edited on almost every RNA observed. Certain embodiments of the invention utilize such high-edit adenosines as result in faster, for example, single-minute time resolutions. In addition, the invention in some aspects includes use of dimerization systems, instead of the constantly-active MCP-MS2 system, to link ADAR or CDAR to constitutively expressed repRNAs in a stimulus-specific manner. In certain aspects of the invention systems are prepared and used to record both age and a stimulus-specific signal on a single repRNA. In addition, the invention includes, in some aspects systems and methods for reporting on timing of other kinds of cellular events such as but not limited to: calcium-related effects and the effect of one or more signaling molecules. The aspects of the invention set forth above, as well as the additional aspects and embodiments set forth elsewhere herein support use of RNA tickertape as a scalable and extensible approach for recording the histories of cells.
Table 1 provides a list of oligonucleotides used in embodiments of RNA-based molecular recording systems set forth herein.
Table 2 provides a list of RNA editing templates used in embodiments of RNA-based molecular recording systems set forth herein.
A new class of molecular recorders based on RNA has now been designed and developed. An RNA-based molecular recorder of the invention, also referred to herein as a “tickertape system” of the invention may be thought of as a temporal microscope, which, rather than mapping point sources onto Airy functions via an objective lens, maps instantaneous transcriptional events onto Poisson binomial editing distributions via the statistical dispersion intrinsic to Poisson processes. Analogous to how fluorescent reporter proteins allow for non-invasive spatial readout of cell state by imaging, reporter RNAs of the invention allow for non-invasive temporal readout of cell state by sequencing. However, whereas the spatial transfer function of a microscope with finite numerical aperture is not invertible, the temporal transfer function between transcription space and editing space is in principle invertible, so the ability of tickertape methods and systems of the invention to infer arbitrary temporal functions of transcriptional activity is not limited by any fundamental diffraction-like limit. Instead, RNA tickertape systems of the invention are limited only by how well the observed editing distribution approximates the true editing distribution, which is a statistical sampling problem. By increasing the number of observations per cell (for example by increasing the number of editing sites per RNA), the accuracy achieved both in bulk and in the single cell case may be made substantially higher.
Embodiments of systems and methods of the invention can be used in cell culture, in vitro preparations, and in in vivo settings. Some aspects of the invention include use of RNA-based molecular reporter systems and components for determining in one or more cells one or more of events such as but not limited to: (i) the timing of transcriptional activity in the cell, and (ii) the presence or absence of an effect resulting from a stimulus received by the cell on transcription in the cell. Embodiments of methods of the invention can be used to record the timings of transcriptional events in real time coordinates in single cells, a plurality of cells, tissues, and organisms.
Embodiments of molecular recorder systems of the invention are capable of recording the timings of transcriptional events in real time coordinates. It has now been determined that the history of the activity of a given promoter can be inferred from the distribution of ages of the RNAs generated by that promoter (
In one embodiment of a system of the invention, reporter RNAs (repRNAs) have been designed and produced that are capable of reporting their age via the gradual accumulation of edits, for example though not intended to be limiting, A→I edits caused by a modified version of the human Adenosine Deaminase Acting on RNA 2 (ADAR2) enzyme (
Previous systems for temporally resolved detection of neural activity in single cells have relied on methods such as optical detection, or the detection of electric or magnetic fields, and, therefore it has been challenging or not possible to record from many neurons simultaneously, or from deep neural populations. Although the time resolution of RNA tickertape is intrinsically limited by the speed of RNA transcription, it has now been demonstrated that tickertape can be used to perform a sequencing-based readout of the history of activity in a population of neurons with temporal resolution comparable to that of immediate early genes, which are popular for detection of neurons recently active in a neural network, but are primarily used to perform such measurements at single time points (19).
The experimental analysis described herein has in part excluded a set of 12 adenosines that were identified as being edited with much higher rate constants than the other adenosines on the tested repRNAs. The 12 adenosines were identified as being edited on almost every RNA observed. Additional aspects of the invention can take advantage of these rapidly edited RNAs to achieve faster resolution, for example, though not intended to be limiting, single-minute time resolutions. In other aspects of the invention an alternative dimerization system may be used, instead of the constantly-active MCP-MS2 system, in order to link an ADAR or CDAR to constitutively expressed repRNAs in a stimulus-specific manner, or to record both age and a stimulus-specific signal on a single repRNA. In some aspects of the invention tickertapes are constructed that can be used to report on the timing of other kinds of cellular events, non-limiting examples of which are: the impact of contact with calcium or signaling molecules. Embodiments of RNA tickertape systems (also referred to herein as RNA-based molecular recorder systems) can be used as a scalable and extendible approach to record and determine the histories of cells.
Methods to prepare and use system components of the invention such as a repRNA, a predetermined enzyme, and fusion proteins are described herein and also may include art-known methods to deliver and express encoded molecules.
Certain aspects of the invention comprise methods for inclusion and use of RNA-based molecular recorders of the invention in one or more cells, which permits detection in one or more cells. Embodiments of RNA-based molecular recorder systems of the invention can be introduced in specific cells (e.g., using a virus, vector, or other means for delivery) and used to assess stimuli that impact RNA transcription in the cells. The cells may be in intact organisms (including humans) as well as cells in vitro, and in cells in culture.
Certain embodiments of RNA-based molecular recorders of the invention include a repRNA and a predetermined enzyme in a cell. Using such a system of the invention permits determination of one or more alterations that take place in the composition of the repRNA. As used herein, the term: “composition of the repRNA” means the sequence components of the repRNA. An alternation in one or more sequence components of a repRNA means an alteration in the nucleic acid sequence or in a feature of one or more of the nucleic acids in the sequence. For example, a change in a nucleic acid sequence may be a nucleotide insertion, deletion, or substitution, (such as but not limited to an A→G substitution). A non-limiting example of an alteration in a feature of a nucleic acid is a modification of the base itself, rather than a change in the sequence per se. It will be understood that an alteration in the composition of a repRNA may include one or more sequence changes and/or a modification of one or more bases in the repRNA sequence. Various types of base modifications are well known the art.
Embodiments of the invention include molecular recording systems as well as components thereof. For example, components of a molecular recording or tickertape system of the invention include but are not limited to, repRNAs and predetermined enzyme compositions. In some embodiments, a repRNA of the invention includes an editing array in the 3′ UTR of a target RNA. As used herein the term “editing array” refers to a sequence array that includes at least one of (i) one or more engineered binding sites; and (ii) one or more selectively favored substrates of the predetermined enzyme. In certain embodiments of methods of the invention, a predetermined enzyme is selected along with its favored substrate to which it specifically binds and the specifically favored substrate is included in an editing array that is part a repRNA. A target RNA may in some aspects of the invention be an exogenous RNA that is designed and delivered into a cell and in certain aspects of the invention a target RNA is an endogenous RNA that naturally occurs in a cell. A target RNA may be an mRNA, a modified RNA, an RNA with one or more sequence or base variations with respect to RNAs described herein.
Non-limiting examples of editing arrays are: adenosine-rich editing array and a cytosine-rich editing array. In some aspects of the invention, a repRNA includes an engineered binding site positioned in an editing array of the repRNA. The engineered binding site may be designed and prepared such that it selectively binds to the predetermined enzyme. In some aspects of the invention, the predetermined enzyme is attached to a polypeptide that specifically binds to the engineered binding site—thus the binding of the attached polypeptide to the engineered binding site effectively attaches (which may also be referred to herein as “binds”) the predetermined enzyme to the engineered binding site.
A number of different engineered binding sites and corresponding binding partner molecule may be used in systems and methods of the invention. For example, though not intended to be limiting, an engineered binding site may be engineered MS2 binding site for which an MS2 Capsid protein (MCP) is its selective binding partner, and wherein the MCP is capable of binding to one of the MS2 binding sites that are engineered into the repRNA editing array. In certain aspects of the invention, an MCP protein may be attached to the predetermined enzyme and the binding of the MCP protein to its selective binding partner—the MS2 binding site—in the editing array of the repRNA molecule, thereby attaching the predetermined enzyme to the array. In certain aspects of the invention, the predetermined enzyme and the selective binding partner for the engineered binding site may be present as part of a fusion protein.
In certain aspects of the invention, a molecular reporter system or component thereof can be expressed in a cell and used to determine characteristics such as, but not limited to the timing of transcriptional events and the effect of stimuli on transcription in the cell. In some embodiments, a baseline determination of one or more characteristics of transcription in a “control” cell can be performed using a system of the invention. Such baseline determinations may be made for the same characteristics that are also determined in similar cells but under different circumstances. For example, a baseline determination may indicate a “control” characteristic which can be compared to the characteristic in a “test” cell that is exposed to one or more different stimuli, environmental changes, etc. to which the control cell was not exposed. For example, though not intended to be limiting, a test cell that includes a repRNA system of the invention can be contacted with a test agent such as a biological agent, etc. and a difference in one or more characteristics in the test cell compared to a control cell not contacted with the biological agent in order to ascertain whether there is an effect of the agent on transcription in the cell. Non-limiting examples of test agents are: a candidate compound, a pharmaceutical compound, an electrical stimulus, a chemical stimulus, a biological stimulus, a signaling molecule, a signaling chemical, a temperature stimulus, and a light stimulus. Additional stimuli and agents that are suitable for use in embodiments of the invention are known and routinely used in the art.
It will be understood that in some aspects of the invention, a stimulus or test agent may be delivered directly to a cell that includes a molecular recorder system of the invention, or may be delivered to another cell that is in communication with a cell that includes a molecular recorder of the invention. As used herein, the term “in communication with” used in reference to a cell that includes an RNA-based molecular recorder of the invention, includes cells, for example, that influence the cell comprising the RNA-based molecular recorder, for example, though not intended to be limiting, via a neurotransmitter means, an electrical means, etc. Communication can be direct communication from a cell immediately (directly) upstream from the cell that includes a molecular recorder system of the invention, or can be indirect communication, such as the result of activity of a cell further (indirectly) upstream that impacts the cell in which a molecular recorder of the invention is included. Stimulation of one or more of a cell directly upstream and a cell indirectly upstream may result in a change in transcription in a cell that includes a molecular recorder of the invention, and the presence of the molecular recorder permits determination of changes in characteristics of transcription in that cell using methods of the invention. As used herein a change in transcription means an alteration in the transcription characteristic, for example an increase in a rate or timing of transcription, a decrease in a rate or timing of transcription, the start of transcription, a delay in the start of transcription, and the like.
Methods and molecular recorder systems of the invention can be used to assess one or more changes in: (1) an internal environment of a cell, (2) an external environment of a cell, (3) an internal environment of an upstream cell, and (4) an external environment of an upstream cell. Non-limiting examples of events and situations that may change in a cell's internal or external environment and that can directly or indirectly effect transcription in a cell comprising a molecular recorder of the invention include, an action potential, a disease or injury condition in the cell or subject comprising the cell, contact of the cell with a test agent or compound, contact of the cell with a pharmaceutical agent or compound, a surgical procedure in the subject, contact of the cell with radiation, light, electric stimulation, etc. Other types of events and actions that alter the internal or external environment of a cell are known in the art, and can also be assessed using methods and RNA-based molecular recorders of the invention.
Components of RNA-based molecular recorder systems of the invention are well suited for targeting cells, expression in cells, and for use to detect and assess transcription levels and changes associated with stimuli and/or cell activities. In some embodiments, a molecular recorder system of the invention can be utilized to detect one or more of conductance changes across cell membranes, the impact of endogenous signaling pathways (such as calcium dependent signaling, etc.), and the effect of applied candidate compounds and agents on a cell that includes the molecular recorder of the invention. Thus, certain aspects of the invention include methods of using RNA-based molecular recorders to screen putative therapeutic agents, known therapeutic agents, combinations of two or more independently selected known and putative therapeutic agents. One or more RNA-based molecular recorders of the invention can also be used in some embodiments of methods of the invention to assess the effect of internal cellular conditions, environmental conditions external to the cell, and to assess the result diseases, injuries, treatments, etc. on transcription in the cell comprising the molecular recorder. Methods and systems of the invention can also be used to examine normal cells in vitro and in vivo. For example, in some embodiments a RNA-based molecular recorder system can be used to determine transcription events in normal cells and subjects and the resulting information on transcription characteristics can be applied in the study of normal cell development, non-limiting examples of which are cell development in regeneration, embryonic cell development, establishment of cell connectivity, and the like.
The present invention, in part, includes novel RNA-based molecular recorder systems and components thereof, their expression in cells, and their use to determine alterations in characteristics of transcription in the host cell. As used herein, the term “host cell” means a cell that includes one or more components of an RNA-based molecular recorder system of the invention. Non-limiting examples of components of molecular recorder systems of the invention are described herein, see for example, Tables 1-3 and the Examples section. Aspects of the invention also include additional functional variants of components of RNA-based molecular recorder systems described herein, including polynucleotides, polypeptides, compositions comprising the components and functional variants thereof, and methods of using the components and functional variants thereof to perform RNA-based molecular recording in a cell., or a plurality of cells. As used herein the term “plurality of cells” means more than one cell, which in some embodiments of the invention is more than 1, more than 10, more than 100, more than 1000, more than 10,000, or more than 100,000, and more than 1,000,000, including all integers within the range from more 1 to more than 1,000,000
It is understood that the terms: RNA-based molecular recorder system components and tickertape system components encompass molecules, polypeptides, and polynucleotides described herein, as well as functional variants thereof. The invention also includes compounds and compositions that comprise one or more components of an RNA-based molecular recorder system of the invention. A compound or composition that comprises a component of a molecular recorder of the invention such as a predetermined enzyme or a repRNA may include only that component, may include both of those components, or may include one, two, three, four, five, six, or more additional elements. Non-limiting examples of additional elements are: a vector, a promoter, a detectable label sequence, a trafficking sequence, a delivery molecule sequence, an additional sequence, etc. The term “RNA-based molecular recorder” is used herein in reference to a repRNA and predetermined enzyme components or encoding molecules.
Certain embodiments of the invention include polynucleotides comprising nucleic acid sequences that encode a component of a molecular recorder system of the invention, and some aspects of the invention comprise methods of delivering and/or using such polynucleotides in cells, tissues, and/or organisms. RNA-based molecular recorder component polynucleotide sequences and amino acid sequences used in aspects and methods of the invention may be “isolated” sequences. As used herein, the term “isolated” used in reference to a polynucleotide, nucleic acid sequence, polypeptide, or amino acid sequence means a polynucleotide, nucleic acid sequence, polypeptide, or amino acid sequence, respectively, that is separate from its native environment and present in sufficient quantity to permit its identification or use. Thus, a nucleic acid or amino acid sequence that makes up a component of an RNA-based molecular recorder molecule that is present in one or more of a vector, a cell, a tissue, an organism, etc., may be considered to be an isolated sequence if it is not naturally present in that cell, tissue, or organism, and/or did not originate in that cell, tissue, or organism.
A host cell means a cell that comprises one or more components of an RNA-based molecular recorder. In certain aspects of the invention one or more components of an RNA-based molecular recorder system of the invention are delivered into and/or expressed in a cell. Examples of a host cells include, but are not limited to vertebrate cells, mammalian cells (including but not limited to non-human primate, human, dog, cat, horse, mouse, rat, etc.), insect cells (including but not limited to Drosophila, etc.), fish, worm, nematode, and avian cells. In some embodiments of the invention a cell is a plant cell.
One or more components of an RNA-based molecular reporter system of the invention may be derived from (also referred to herein as “being a variant of”) one or more components disclosed herein, and they may exhibit the same qualitative function and/or characteristics of the molecular reporter system component from which they have been derived, and/or may show one or more increased or decreased level of a function or characteristic of the parent component. In some embodiments of the invention an effectiveness of a variant or derived component of a molecular reporter system set forth herein may differ from the parent component. For example, in some instances a variant or derived component is capable of faster determination of a characteristic of transcription in a host cell than is possible for its parent component.
It is understood in the art that the codon systems in different organisms can be slightly different, and that therefore where the expression of a given protein from a given organism is desired, the nucleic acid sequence can be modified for expression within that organism. Thus, in some embodiments, a polynucleotide that encodes a component of an RNA-based molecular recorder system of the invention comprises a mammalian-codon-optimized nucleic acid sequence, which may in some embodiments be a human-codon optimized nucleic acid sequence. Codon-optimized sequences can be prepared using routine methods.
Delivery of one or more components of an RNA-based molecular recorder of the invention to a cell and/or expression of the component in a cell can be done using art-known delivery means. [see for example, Chow et al. Nature 2010 Jan. 7;463(7277):98-102; and for Adeno-associated virus injection: Betley, J. N. & Sternson, S. M. (2011) Hum. Gene Ther. 22, 669-677; for In utero electroporation: Saito, T. & Nakatsuji, N. (2001) Dev. Biol. 240, 237-46; for microinjection into zebrafish embryos: Rosen J. N. et al., (2009) J. Vis. Exp. (25), e1115, doi:10.3791/1115; and for DNA transfection for neuronal culture: Zeitelhofer, M. et al., (2007) Nature Protocols 2, 1692-1704, the content of each of which is incorporated by reference herein in its entirety].
In some embodiments of the invention a component of an RNA-based molecular recorder of the invention is included as part of a fusion protein. It is well known in the art how to encode, prepare, and utilize fusion proteins that comprise a polypeptide sequence. In certain embodiments of the invention, a vector that encodes a fusion protein can be prepared and used to deliver a component of an RNA-based molecular recorder system of the invention to a cell and can also in some embodiments be used to target delivery of a component of an RNA-based molecular recorder system of the invention to a specific cell, cell type, tissue, or region in a subject. Suitable targeting sequences useful to deliver a component of an RNA-based molecular recorder of the invention to a cell, tissue, region of interest are known in the art. Delivery of a component of an RNA-based molecular recorder system of the invention to a cell, tissue, or region in a subject can be performed using art-known procedures. A fusion protein of the invention can be delivered to a cell by delivery of a vector encoding a fusion protein. The delivered fusion protein is then expressed in a specific cell type, tissue type, organ type, and/or region in a subject, or in vitro, for example in culture, in a slice preparation, etc.
In certain aspects of the invention, a component of an RNA-based molecular recorder system of the invention is non-toxic or substantially non-toxic to the cell into which it is delivered and/or expressed. In some embodiments of the invention, a component of an RNA-based molecular recorder of the invention is genetically introduced into a cell, and reagents and methods are provided for genetically targeted expression of components of an RNA-based molecular recorder system of the invention. Genetic targeting can be used to deliver one or more components of an RNA-based molecular recorder system of the invention to specific cell types, to specific cell subtypes, to specific spatial regions within an organism. In some embodiments of the invention, targeting can be used to control of the amount of a component of an RNA-based molecular recorder system of the invention that is expressed and the timing of the expression. Preparation, delivery, and use of a fusion protein and its encoding nucleic acid sequences are well known in the art. Routine methods can be used in conjunction with teaching herein to express one or more RNA-based molecular recorder system components and optionally additional polypeptides, in a desired cell, tissue, or region in vitro or in a subject.
Some embodiments of the invention include a reagent for genetically targeted expression of a component of an RNA-based molecular recorder system of the invention, wherein the reagent comprises a vector that contains the gene for the component. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting between different genetic environments another nucleic acid to which it has been operatively linked. The term “vector” may also refer to a virus or organism that is capable of transporting the nucleic acid molecule. One type of vector is an episome, i.e., a nucleic acid molecule capable of extra-chromosomal replication. Some useful vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors.” Other useful vectors, include, but are not limited to viruses such as lentiviruses, retroviruses, adenoviruses, and phages. Vectors useful in some methods of the invention can genetically insert an RNA-based molecular recorder system of the invention into dividing and non-dividing cells and can insert an RNA-based molecular recorder system of the invention into an in vivo, in vitro, or ex vivo cell.
Vectors useful in methods of the invention may include additional sequences including, but not limited to one or more signal sequences and/or promoter sequences, or a combination thereof. Expression vectors and methods of their use are well known in the art. Non-limiting examples of suitable expression vectors and methods for their use are provided herein. In certain embodiments of the invention, a vector may be a lentivirus comprising the gene for an RNA-based molecular recorder system of the invention. A lentivirus is a non-limiting example of a vector that may be used to create stable cell line. The term “cell line” as used herein is an established cell culture that will continue to proliferate given the appropriate medium.
Promoters that may be used in methods and vectors of the invention include, but are not limited to, cell-specific promoters or general promoters. Methods for selecting and using cell-specific promoters and general promoters are well known in the art. A non-limiting example of a general purpose promoter that allows expression of an RNA-based molecular recorder system of the invention in a wide variety of cell types—thus a promoter for a gene that is widely expressed in a variety of cell types, for example a “housekeeping gene” can be used to express RNA-based molecular recorder system component(s) of the invention in a variety of cell types. Non-limiting examples of general promoters are provided elsewhere herein and suitable alternative promoters are well known in the art. In certain embodiments of the invention, a promoter may be an inducible promoter, examples of which include, but are not limited to tetracycline-on or tetracycline-off, or tamoxifen-inducible Cre-ER.
In some embodiments of the invention a reagent for expression of a component of an RNA-based molecular recorder system of the invention is a vector that comprises a gene encoding the component, and optionally a gene encoding one or more additional polypeptides. Vectors useful in methods of the invention may include additional sequences including, but not limited to, one or more signal sequences and/or promoter sequences, or a combination thereof. In certain embodiments of the invention, a vector may be a lentivirus, adenovirus, adeno-associated virus, or other vector that comprises a gene encoding RNA-based molecular recorder system component(s) of the invention. An adeno-associated virus (AAV) such as AAV8, AAV1, AAV2, AAV4, AAV5, AAV9, are non-limiting examples of vectors that may be used to express a fusion protein of the invention in a cell and/or subject. Expression vectors and methods of their preparation and use are well known in the art. Non-limiting examples of suitable expression vectors and methods for their use are provided herein. Other vectors that may be used in certain embodiments of the invention are provided in the Examples section herein.
Promoters that may be used in methods and vectors of the invention include, but are not limited to, cell-specific promoters or general promoters. A non-limiting examples promoters that can be used in vectors of the invention are: ubiquitous promoters, such as, but not limited to: CMV, CAG, CBA, and EF1a promoters; and tissue-specific promoters, such as but not limited to: Synapsin, CamKIIa, GFAP, RPE, ALB, TBG, MBP, MCK, TNT, and aMHC promoters. Methods to select and use ubiquitous promoters and tissue-specific promoters are well known in the art. A non-limiting example of a tissue-specific promoter that can be used to express a component of an RNA-based molecular recorder system of the invention in a cell such as a neuron is a synapsin promoter, which can be used to express the component in certain embodiments of methods of the invention. Additional tissue-specific promoters and general promoters are well known in the art and, in addition to those provided herein, may be suitable for use in compositions and methods of the invention. Other non-limiting examples of promoters that may be used in certain embodiments of methods of the invention are provided in the Examples section.
Additional molecules that can be administered and delivered to a cell in a method or system of the invention, include, but are not limited to: opsin polypeptides, detectable label polypeptides, fluorescent polypeptides, additional trafficking polypeptides, etc.
Non-limiting examples of detectable label polypeptides that may be included in a composition comprising a component of an RNA-based molecular recorder system of the invention are: green fluorescent protein (GFP); enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP); yellow fluorescent protein (YFP), dtTomato, mCardinal, mCherry, DsRed, cyan fluorescent protein (CFP); far red fluorescent proteins, etc. Numerous fluorescent proteins and their encoding nucleic acid sequences are known in the art and routine methods can be used to include such sequences in fusion proteins and vectors, respectively, of the invention.
Additional sequences that may be included in a fusion protein comprising a component of an RNA-based molecular recorder system of the invention are trafficking sequences, including, but not limited to: Kir2.1 sequences and functional variants thereof, KGC sequences, ER2 sequences, etc. Trafficking polypeptides and their encoding nucleic acid sequences are known in the art and routine methods can be used to include and use such sequences in fusion proteins and vectors, respectively, of the invention.
Table 3 provides a list of plasmids that have been prepared and used in RNA-based molecular recorder systems and components of the invention. Addition plasmids may also be used, for example pCMV Tet3G (Clontech) has also been used in embodiments of the invention. Those skilled in the art will be able to prepare additional suitable plasmids using routine methods in conjunction with information provided herein.
Some aspects of the invention include cells used in conjunction with an RNA-based molecular recorder system of the invention. Cells in which an RNA-based molecular recorder system component may be expressed, and that can be used in methods of the invention, include prokaryotic and eukaryotic cells. Certain embodiments of the invention, include use of mammalian cells; including but not limited to cells of humans, non-human primates, dogs, cats, horses, rodents, etc. In some embodiments of the invention, cells that are used are non-mammalian cells; including but not limited to insect cells, avian cells, fish cells, plant cells, etc. An RNA-based molecular recorder system of the invention may be included in non-excitable cells and in excitable cells, the latter of which include cells able to produce and respond to electrical signals. Examples of excitable cell types include, but are not limited, to neurons, muscle cells, visual system cells, sensory cells, auditory cells, cardiac cells, and secretory cells (such as pancreatic cells, adrenal medulla cells, pituitary cells, etc.), cardiac cells, immune system cells, etc.
Cells in which an RNA-based molecular recorder system of the invention can be used include embryonic cells, stem cells, pluripotent cells, mature cells, geriatric cells, as well as cells in other developmental stages. Non-limiting examples of cells that may be used in methods of the invention include: neuronal cells, nervous system cells, cardiac cells, circulatory system cells, kidney cells, liver cells, epiderminal cells, visual system cells, auditory system cells, secretory cells, endocrine cells, and muscle cells.
In some embodiments, a cell used in conjunction with methods and an RNA-based molecular recorder system of the invention is a healthy normal cell that is not known or suspected of having a disease, disorder, or abnormal condition. In some embodiments of the invention, a cell used in conjunction with methods and an RNA-based molecular recorder system of the invention may in some embodiments be a normal cell or in some embodiments is an abnormal cell. Non limiting examples of elements of an abnormal cell are: (1) a cell that has a disorder, disease, or condition; (2) a cell obtained from a subject that has, had, or is suspected of having disorder, disease, or condition; (3) a cell known to be or suspected of being involved in a disorder, disease, or condition; and (4) a cell that is a model for a disorder, disease, or condition, etc. Non-limiting examples of such cells are: a degenerative cell, a neurological disease-bearing cell, a cell model of a disease or condition, an injured cell, a cell downstream from a disease-bearing or injured cell, etc. In some embodiments of the invention, a cell may be a control cell. A cell that is directly or indirectly upstream from a cell in which an RNA-based molecular recorder system may be included may be a normal cell or may be an abnormal cell.
An embodiment of an RNA-based molecular recorder system of the invention may be included in a cell from or in culture, a cell in solution, a cell obtained from a subject, and/or a cell in a subject (in vivo cell). In some embodiments of the invention, an RNA-based molecular recorder system is present in and monitored in cultured cells, cultured tissues (e.g., brain slice preparations, etc.), and in living subjects, etc. As used herein, a the term “subject” may refer to a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat, bird, rodent, fish, insect, or other vertebrate or invertebrate organism. In certain embodiments, a subject is a mammal and in certain embodiments a subject is a human. Additional non-limiting examples of cell types that may be used in certain methods of the invention are provided in the Examples section, as are non-limiting examples of organisms that may subjected to certain methods of the invention.
A cell that includes an RNA-based molecular recorder system and/or component of the invention may be a single cell, an isolated cell, a cell in culture, an in vitro cell, an in vivo cell, an ex vivo cell, a cell in a tissue, a cell in a subject, a cell in an organ, a cell in a cultured tissue, a cell in a neural network, a cell in a brain slice, a neuron, a cell that is one of a plurality of cells, a cell that is one in a network of two or more interconnected cells, a cell in communication with another cell, a cell that is one of two or more cells that are in physical contact with each other, etc. It will be understood that methods of the invention can be carried out in a plurality of cells such that one or more cells comprises the RNA based molecular recorder system of the invention. Inclusion of a system of the invention in a plurality of cells permits monitoring and determining one or more alterations in the composition of a repRNA across the plurality of cells.
An RNA-based molecular recorder system of the invention and methods of using such molecular recorder systems can be utilized to assess changes in cells, tissues, and subjects in which the system is included. Some embodiments of the invention include use of an RNA-based molecular recorder system of the invention to identify effects of candidate compounds on cells, tissues, and subjects. Results of testing cell transcription activity using an RNA-based molecular recorder of the invention can be advantageously compared to a control. In some embodiments of the invention an RNA-based molecular recorder system may be in a cell or cell population and used to test the effect of candidate compounds on the cell or population, respectively. A “test” cell, tissue, or organism may be a cell, tissue, or organism in which activity of an RNA-based molecular recorder system of the invention can be determined or assayed. Results obtained using assays and tests of a test cell, tissue, or organism may be compared with results obtained from the assays and tests performed in other test cells, tissues, or organisms or assays and tests performed in control cells, tissues, or organisms.
As used herein a control value may be a predetermined value, which can take a variety of forms. It can be a single cut-off value, such as a median or mean. It can be established based upon comparative groups, such as cells or tissues that include an RNA-based molecular recorder system of the invention that is under essentially the same conditions of test cells but are not contacted with a candidate compound. Another non-limiting example of a comparative group includes cells or tissues that have a disorder or condition and groups without the disorder or condition. Another non-limiting example of comparative group includes cells from a subject or subjects with a family history of a disease or condition and cells from a subject or subjects without such a family history. A predetermined value can be arranged, for example, where a tested population is divided equally (or unequally) into groups based on results of testing. Those skilled in the art are able to select appropriate control groups and values for use in comparative methods of the invention.
Administration of a component of an RNA-based molecular recorder system of the invention may include, but is not limited to: administering to a cell or subject a composition that includes a vector comprising a polynucleotide sequence that encodes the component, administering to a cell or subject a composition comprising the component, and administering to a subject a cell in which the component is present. A composition of the invention optionally includes a carrier, which may be a pharmaceutically acceptable carrier.
A component of an RNA-based molecular recorder system of the invention may be administered to a cell and/or subject in a formulation, which may be administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally additional ingredients. In some aspects, a pharmaceutical composition comprises one or more RNA-based molecular recorder system component(s) of the invention and a pharmaceutically-acceptable carrier. Pharmaceutically acceptable carriers are well known to the skilled artisan and may be selected and utilized using routine methods. As used herein, a pharmaceutically acceptable carrier means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Pharmaceutically acceptable carriers may include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials that are well-known in the art. Exemplary pharmaceutically acceptable carriers are described in U.S. Pat. No. 5,211,657 and others are known by those in the art.
The terms “delivery into” and “include” when used herein to describe an action that results in a component of an RNA-based molecular recorder system of the invention being present in a cell, are intended to encompass delivery of the component(s) into the cell (for example, though not intended to be limiting, in the form of a fusion protein), and delivery of a polynucleotide sequence that encodes the component and that is subsequently expressed in the cell. A component of an RNA-based molecular recorder system of the invention may be administered using art-known methods. The absolute amount to be delivered can be determined using routine methods. The delivery may be done in a single administration, a single or multiple deliveries, and if delivered into a subject may be based on individual subject parameters including age, physical condition, size, weight, and the stage of a disease or condition, test parameters to be followed, etc. These factors can be addressed with no more than routine experimentation.
Various modes of administration will be known to one of ordinary skill in the art that can be used to effectively deliver one or more components of an RNA-based molecular recorder system of the invention in a desired cell, tissue, cell of a subject, organ of a subject, or region of a subject. Methods for administering a composition comprising a component of an RNA-based molecular recorder system of the invention may include, but are not limited to: injection, microinjection, perfusion, electroporation, or other suitable means. The invention is not limited by the particular modes of administration disclosed herein and additional art-known delivery means may be suitable for administration of components of an RNA-based molecular recorder system of the invention.
Other protocols suitable for administration of one or more components that are part of an RNA-based molecular recorder system of the invention are known to those in the art. Embodiments of methods of the invention to administer a cell or vector to increase a level of a component of an RNA-based molecular recorder system of the invention in an animal other than a human; and administration and use of an RNA-based molecular recorder system of the invention for testing purposes or veterinary purposes, are substantially the same as described above. It will be understood by a skilled artisan that this invention is applicable to both human and animals.
Disorders, conditions, and events that may be assessed using methods of the invention to include an RNA-based molecular recorder of the invention in a cell, tissue, and/or subject and to use the system to determine characteristics of transcription in the cell. Methods and systems of the invention may be used to assess early stage development, cell and tissue regeneration, cell communication, disease, etc. Diseases that may be examined using methods and systems of the invention include, but are not limited to: injury, brain damage, spinal cord injury, epilepsy, metabolic disorders, cardiac dysfunction, vision loss, blindness, deafness, hearing loss, and neurological conditions (e.g., Parkinson's disease, Alzheimer's disease, and seizure), degenerative neurological conditions, drug contact, toxins, etc. In some embodiments of the invention, a disorder or condition may be monitored by including an RNA-based molecular recorder system of the invention in at least one cell and monitoring characteristics of transcription in the cells using the molecular recorder system. In some embodiments of the invention, such methods can be used in methods such as, but not limited to, assessing therapeutic agents and treatments, assessing putative therapeutic agents and treatments, expanding understanding of connectivity between cells, and exploring transcription activity patterns in a cell or cells. An RNA-based molecular recorder system of the invention may be targeted to cells and used to monitor transcription changes in such cells.
The present invention in some aspects, includes one or more of preparing nucleic acid sequences that encode one or more components of an RNA-based molecular recorder system of the invention, expressing in cells one or more components of an RNA-based molecular recorder system encoded by the prepared nucleic acid sequences; activating a promoter in the RNA-based molecular recorder system that activations the repRNAs in the system, and monitoring changes in transcription in the cell by assessing changes in editing of the composition of the repRNA The ability to specifically, consistently, reproducibly, and sensitively monitor changes in the repRNA composition using methods such as sequencing and single cell sequencing has been demonstrated. The present invention enables monitoring of transcription changes in in vivo, ex vivo, and in vitro, and the RNA-based molecular recorder system and its use have broad-ranging applications for drug screening, disease assessment, treatment assessment, and research applications, some of which are describe herein.
All plasmids were constructed either using restriction cloning using restriction enzymes from New England Biosciences and the NEB Quick Ligation kit (M2200L), or using the In-Fusion HD cloning enzyme mix (Clontech, 638911). Plasmids were grown in E.Cloni 10G Chemically Competent Cells (Lucigen, 60107-1) and were verified by Sanger sequencing (Eton biosciences). All plasmids are deposited on Addgene.
Due to high repetition present in the RNA editing templates, inserts for plasmids 76, 147, 148, 149, and 187 (see Table 1) were ordered as sense and antisense ultramer oligonucleotides, which were annealed to each other prior to cloning. Plasmid 76 was cloned by inserting RNA templates (A_Short, B_Short, C, D, E) into the 3′ UTR of an iRFP transcript expressed under a UbC promoter in a second generation lentivirus backbone using SphI and Clal. Subsequently, this plasmid was modified by the addition of a flavivirus xrRNA in the 5′ UTR. Templates A_Short and B_Short were then extended by inserting another pair of annealed ultramers on the 5′ side of A_Short and B_Short using SphI and MluI. The resulting templates are designated A and B. To generate plasmids 147, 148, 149, and 183 (used in certain experiments herein), templates A and B were then moved into different backbones and different promoters by restriction cloning, or by Gibson assembly with PCR amplification of the repRNA template region. Template A is used throughout the Examples, and Template B is shown in
All cell cultures were lysed with 600μL of buffer RLT Plus from the Qiagen RNEasy Plus Mini Kit (Qiagen, 74136), and were pipetted up and down vigorously to homogenize. RNA was then purified using the Qiagen RNEasy Plus Mini kit, following the instructions from the manufacturer. Subsequently, 11 μL of purified RNA was reverse transcribed using Superscript IV (Thermofisher, 18090050) and a barcoded version of SGR-174 (see Table 1), following the protocol from the manufacturer. Reverse transcription reactions were then purified using Agencourt Ampure XP beads at a 1:1 dilution (Beckman-Coulter, A63881). Some portion of the eluent, typically 25%, was then PCRed using P5 and a barcoded version of SGR-176 (see Table 1) the Q5 Hot Start High Fidelity 2× Master Mix (NEB, M0492L) with the following settings: 30 s of 98° C. denaturation; then 25-30 cycles of 10 s denaturation at 98° C., 20 s annealing at 70° C., and then 25 s extension at 72° C. Neuron lysates were typically PCRed for 30 cycles, while HEK cell lysates were typically PCRed for 25 cycles. PCR reactions were then pooled and run on a gel, and a 400 bp band was extracted using the NucleoSpin PCR Cleanup Kit (Macherey-Nagel, 740609.250). The concentration of DNA in the resulting eluent was determined via a Qubit 2 fluorometer (Thermofisher), and was then adjusted to 4 nM for sequencing. The read structure is shown in
Sequencing was performed using NextSeq Mid Output 300 cycle kit (Illumina, FC-404-2004) or Miseq 300 cycle v2 kits (MS-102-2002), with at least 80 bp read 1 and 185 bp read 2, with 8 bp index 1 and 15 bp index 2.
Except in the case of the single cell experiments, HEK293FT and 3T3 cells were plated in 24 well plates. Cells were grown in DMEM (Thermofisher, 10566016), supplemented with Penicillin/Streptomycin (Thermofisher, 15140122) and 10% certified Tet-system approved FBS (Clontech, 631101). Transfections were performed using the TransIT-X2 system (Mirus, MIR 6000), following the manufacturer's instructions.
For doxycycline experiments, HEK and 3T3 cells in 24 well plates were transfected with 300 ng of plasmid 147 or 148, 100 ng of pCMV Tet3G from the Tet-on 3G system (Clontech, 631168), and 100 ng of plasmids 116v1, 116v5, or 116v6. In the experiments with results shown in
For experiments using the Vivid promoter, 3T3s were transfected with 300ng of plasmid 149, 100 ng of pCMV Tet3G, and 100 ng of plasmid 116v5. For conditions in which cells were transfected with both plasmid 147 and plasmid 149, they received 150 ng of each plasmid. For the experiments in
For the experiment with results shown in
For the experiment in
For all experiments involving single cells, HEK cell cultures were prepared, transfected with 100 ng of pAAV-CAG-GFP (Addgene 37825), 200 ng of plasmid 147, 100 ng of plasmid 116v5, and 100 ng of pCMV Tet3G, stimulated with doxycycline, and then silenced with actinomycin D as described above. Subsequently, at the designated time point (e.g., 8 hours or 4 hours after doxycycline was added to the culture medium), cells were treated with trypsin (Life Technologies, 25300054). Following trypsinization, cells were centrifuged at 850 g, washed in cold PBS, and then resuspended in cold PBS. 96 well plates were prepared, with each well containing a solution of 0.2% Triton-X with 2U/μL RNAse inhibitor. Individual cells were sorted into the wells of this wellplate using a Moflo Astrios EQ flow cytometer. Following sorting, the wellplate was sealed, centrifuged, and then placed at −80° C. overnight.
The single cell analysis was nominally conducted with cells from 4 hr and 8 hr time points. However, following trypsinization, cells remained in cold PBS for up to an hour and a half due to latencies in the sorting process. For this reason, estimates from the single cells were compared to the estimates for populations of ˜100,000 of the same cells (i.e., stored in cold PBS for the same amount of time) lysed immediately after sorting.
Library preparation for the single cells proceeded as follows. Plates containing single cells were thawed, and 7 μL of nuclease free water was added to the single cells to bring the total volume up to 11 μL. Subsequently, reverse transcription was performed using Superscript IV and the SGR-174 RT primers, as in the case of the bulk samples, with the following modifications. RT primers were distributed so that each cell at a given time point received an RT primer with a different barcode. In addition, for each time point, two no-template RT reactions were performed. Finally, after the 50° C. step in the Superscript IV protocol, the samples were cooled to 37° C. and 20U of Exonuclease 1 (NEB, M0293S) was added to the reaction to remove excess primers. Samples then remained at 37° C. for 10 minutes, before proceeding to the 80° C. heat inactivation step. Following reverse transcription, the RT reactions for all cells and the two no-template controls at a given time point were pooled, cleaned with Ampure XP beads at a 1:1 dilution, and were then PCRed using the same protocol as for the bulk samples. Cells were pooled prior to PCR as a way of reducing the number of cycles necessary to achieve amplification. In order to minimize barcode swapping between cells during the pooled PCR reaction, cells were excluded if they received fewer than 4 times the number of reads that the no-template controls received. In practice, this corresponded to a minimum of roughly 250 reads per cell.
All procedures involving animals at MIT were conducted in accordance with the US National Institutes of Health Guide tier the Care and Use of Laboratory Animals arid approved by the -Massachusetts Institute of Technology Committee on Animal Care. Primary hippocampal neuron culture was prepared as previously described. Neuron cultures were transfected at 6-7 DIV using a commercial calcium-phosphate kit (Thermofisher, K278001), as previously described. Briefly, neurons were transfected with 60Ong of pU-C19, 200ng of plasmid 116v5, and 200 ng of plasmid 187. Neurons were then incubated with calcium-phosphate precipitates for 30-60 minutes, followed by washing with MEM buffer at pH 6.7-6,8 to remove residual precipitates.
Neurons were stimulated at 14-15DIV. Neurons were placed in 1 mL of plating medium (500 mL MEM, 2.5 g glucose, 50 mg transferrin, 1.1 g HEPES, 5 mL 200 mM L-Glutamine, 12.5 mg insulin, 50 mL HI FBS, 10 mL B27 supplement). To stimulate the neurons, 250 μL of 5× depolarization medium was added and the mixture was agitated gently. Neurons were then left for one hour in an incubator. Subsequently, the medium was aspirated and neurons were washed twice in plating medium. They were then left in plating medium for a variable amount of time, before being lysed in 600 μL of buffer RLT Plus.
1. 500 mL MEM (Thermofisher, 51200-038)
2. 2.5 g glucose (Sigma Aldrich, G7528-1KG)
3. 50 mg transferrin (Sigma Aldrich, T1283-500 mg)
4. 1.1 g HEPES (Sigma Aldrich, H3375-500 G)
5. 5 mL 200mM L-Glutamine (Thermofisher, 25030-081)
6. 12.5 mg insulin (Millipore, 407709)
7. 50 mL HI FBS (VWR, 45000-736)
8. 10 mL B27 Supplement (Thermofisher, 17504-044)
1. 170 mM KCl
2. 10 mM HEPES pH 7.4
3. 1 mM MgCl2
4. 2 mM CaCl2
Due to the limited availability of neuron culture at any given time, the data for
The breakdown of the data in
Experiments for
The alignment and analysis pipeline for sequencing data is summarized in
Finally, except as stated in
In
The exponential model in
y
i(t)=1−e−λ
To more accurately capture the experimental setup, yi was modeled as an underlying process which is exponential, but with start time uniformly distributed in [0, tstop], where t=0 represented when doxycycline was added to the cells and tstop was the time at which actinomycin D was added to the cells. Specifically, a function of the form was fit
where tstop was 1 hr and λi was fit to the data using non-linear least squares. This function was fit for times t≥1.5 hr, because the editing distributions for earlier time points are strongly affected by populations of RNA present prior to doxycycline addition (for example, the mean editing rate in
Here, A is a binary vector with each entry corresponding to a specific adenosine in the repRNA editing region. Ak=1 if adenosine k has been edited to inosine, and sum(A) counts the total number of edits in A. Time estimates using the exponential model were then made by minimizing the Kullback-Leibler divergence between p(n,t) and the empirical distribution q(n) over t. p(n,t) was calculated in practice via a dynamic programming approach.
For
The gradient descent in
Note that for the analysis in
To assess the ability of an embodiment of an RNA-based molecular recorder system of the invention to report the timing of transcriptional activity, experiments were performed in which HEK293T cells expressing the RNA tickertape system were incubated under the control of the tetracycline response element (TRE) in medium containing doxycycline for one hour. Actinomycin D, which blocks RNA transcription by binding to DNA (
To assess and determine the timing of events in the TRE-tickertape system, a statistical model was designed that permits prediction of the RNA age distribution as a function of time since doxycycline induction. If the adenosines on the repRNA template are edited independently and uniformly in time, then for each adenosine on the repRNA, the fraction of RNAs with adenosines at that site should decrease exponentially with the time since transcription, with a site-specific rate constant that depends on the local sequence context. For each adenosine on the repRNA, an exponential cumulative distribution function (CDF) was fitted to the editing fraction over time at that base (
The Poisson binomial approach is a useful approach for estimation because it accounts for the exponential nonlinearity inherent in Poisson processes. However, it was also determined that a simple linear interpolation of the mean yielded accurate estimations in many cases. In the case of the TRE tickertape, the mean interpolation estimated the 2.5 hr and 4.5 hr time points as 2.53 hr±0.08 hr and 4.38 hr±0.02 hr (mean±s.d., N=3 replicates), with errors of 5 min±0.3 min and 7.5 min±1.1 min (mean±s.d., N=3 replicates), respectively.
To confirm that the accuracy of the RNA-based molecular recording system was not limited to the TRE tickertape or to HEK cells, similar experiments were performed in 3T3 cells using repRNAs expressed under a light-inducible Vivid promoter, induced with blue light for one hour (17, 18). The timing of light induction was estimated by interpolation of the mean number of edits per RNA, and yielded a temporal resolution of 17.7±7.5 minutes (
The accuracy of RNA tickertape depends on observing enough repRNAs that the empirical distribution of edits per repRNA accurately approximates the true distribution. Because individual cells may express thousands of copies of an mRNA, it was predicted that RNA tickertape is capable of accurate temporal predictions in single cells. Single HEK cells transfected with the TRE tickertape, induced with doxycycline, and then silenced with actinomycin D, were sorted into individual wells of a 96 well plate, followed by single-cell repRNA sequencing (
Having demonstrated the ability to detect the timing of one-hour transcriptional bursts, studies were performed to determine whether RNA tickertape is capable of decoding the time-course of arbitrary transcriptional programs, which was a much more challenging problem. It was determined that arbitrary transcriptional programs could be represented as convex weighted sums of the single-hour editing distributions (i.e., the one-hour “basis distributions”) as measured with the TRE tickertape (
The gradient descent algorithm correctly approximated the weights of simulated distributions to within approximately 60% of the true values (
Finally, to test whether the gradient descent algorithm is effective for decoding empirical editing distributions, studies were performed in which cells were stimulated with doxycycline for 3 or 6 hours, and applied the algorithm to the resulting empirical editing histograms (
In certain experiments the repRNA expression was placed under the control of a c-fos promoter, and the tickertape system was transfected into primary mouse hippocampal neuron culture at 6 days in vitro (DIV), which is popular as a model for the study of coupling between excitation and transcription in neurons (20-21). At 14-15 DIV, neural activity was induced by adding a potassium-based depolarization medium to the culture (see Methods) (
To estimate the temporal history of neural activity, standards were generated by inducing neurons for one hour with the depolarization medium, which were washed back into normal (non-depolarizating) medium, and lysed at one hour intervals. For up to 7 hours after induction, a population of new repRNAs could be seen to gradually accumulate edits. Even in the presence of a large population of background repRNAs generated by constitutively fos+neurons, the mean number of edits per RNA increased linearly over time (
A primary challenge in the detection of neural activity using tickertape is the presence of a large number of fos+ cells at baseline in primary hippocampal neuron culture. For this reason, tickertape applied to the readout of individual neuron activity, or to targeted populations of neurons outperforms the bulk measurements as described.
1. S. D. Perli et al., Continuous genetic recording with self-targeting CRISPR-Cas in human cells. Science. 353, 339-342 (2016).
2. F. Farzadfard, N. Gharaei, Y. Higashikuni, G. Jung, Single-Nucleotide-Resolution Computing and Memory in Living Cells. bioRxiv (2018).
3. F. Farzadfard, T. K. Lu, Genomically encoded analog memory with precise in vivo dna writing in living cell populations. Science. 346 (2014), doi:10.1126/science.1256272.
4. R. Kalhor et al., Rapidly evolving homing CRISPR barcodes. Nat. Methods. 14, 195-200 (2017).
5. R. U. Sheth, S. S. Yim, F. L. Wu, H. H. Wang, Multiplex recording of cellular events over time on CRISPR biological tape. Science. 358 (2017), doi:10.1126/science.aao0958.
6. W. Tang, D. R. Liu, Rewritable multi-event analog recording in bacterial and mammalian cells. Science. 360 (2018).
7. A. Shur, R. M. Murray, Proof of concept continuous event logging in living cells. bioRxiv (2018).
8. K. L. Frieda et al., Synthetic recording and in situ readout of lineage information in single cells. Nature. 541, 107-111 (2016).
9. S. L. Shipman et al., Molecular recordings by directed CRISPR spacer acquisition. Science. 353 (2016), doi:10.1126/science.aaf1175.
10. B. M. Zamft et al., Measuring cation dependent DNA polymerase fidelity landscapes by deep sequencing. PLoS One. 7 (2012), doi:10.1371/journal.pone.0043876.
11. D. Zenklusen, D. R. Larson, R. H. Singer, Single-RNA counting reveals alternative modes of gene expression in yeast. Nat. Struct. Mol. Biol. 15, 1263-1271 (2008).
12. K. D. Piatkevich et al., A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters. Nat. Chem. Biol. 14 (2018), doi:10.1038/s41589-018-0004-9.
13. M. M. Matthews et al., Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity. Nat. Struct. Mol. Biol. 23, 426-433 (2016).
14. A. Kuttan, B. L. Bass, Mechanistic insights into editing-site specificity of ADARs. Proc. Natl. Acad. Sci. 109, E3295-E3304 (2012).
15. T. Eifler, S. Pokharel, P. A. Beal, RNA-seq analysis identifies a novel set of editing substrates for human ADAR2 present in saccharomyces cerevisiae. Biochemistry. 52, 7857-7869 (2013).
16. E. Bertrand et al., Localization of ASH1 mRNA Particles in Living Yeast. Mol. Cell. 2, 437-445 (1998).
17. X. Wang, X. Chen, Y. Yang, Spatiotemporal control of gene expression by a light-switchable transgene system. Nat. Methods. 9, 266-271 (2012).
18. Z. Ma, Z. Du, X. Chen, X. Wang, Y. Yang, Fine tuning the LightOn light-switchable transgene expression system. Biochem. Biophys. Res. Commun. 440, 419-423 (2013).
19. A. H. Marblestone et al., Physical principles for scalable neural recording. Front. Comput. Neurosci. 7,137 (2013).
20. A. E. West et al., Calcium regulation of neuronal gene expression. Proc. Natl. Acad. Sci. U S. A. 98, 11024-31 (2001).
21. A. E. West, E. C. Griffith, M. E. Greenberg, Regulation of transcription factors by neuronal activity. Nat. Rev. Neurosci. 3, 921-931 (2002).
It is to be understood that the methods, compositions, and apparatus which have been described above are merely illustrative applications of the principles of the invention.
Numerous modifications may be made by those skilled in the art without departing from the scope of the invention. Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose and variations can be made by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims. The contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference in their entirety.
This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional application Ser. No. 62/698490 filed Jul. 16, 2018, the disclosure of which is incorporated by reference herein in its entirety.
This invention was made with government support under grants 1DP5OD024583, 1R01MH114031 6937063, 1RM1HG008525 6932903, 2R01DA029639 6932279, 1R01MH103910 6928799, and 1DP1NS087724 6928706 from the National Institutes of Health and grant W911NF1510548 6933228 from the US Army Research Office. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US19/41962 | 7/16/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62698490 | Jul 2018 | US |
