Use of homology direct repair to record timing of a molecular event

A log of molecular events experienced by a cell and timing indicators for those events are stored in existing polynucleotides through a process of creating a double strand break (“DSB”) in a polynucleotide and inserting a new polynucleotide sequence by repairing the DSB with homology directed repair (“HDR”). The presence, order, and number of new polynucleotide sequences provides a log of events and timing of those events. Cellular mechanisms for creating the DSB and/or repairing with HDR are regulated by intra- or extra-cellular signals. When the log is created in the DNA of a cell, the changes may be heritably passed to subsequent generations of the cell. A correlation between the cellular signals and sequence of inserted HDR templates allows for identification of events and the timing experienced by the cell.

BACKGROUND

The ability to sense and record information through microscopic, inexpensive and easy-to-fabricate devices (i.e., cells) has dramatic potential for numerous sensing applications including diagnostic health measurements within an organism or sensing physical phenomena on a larger scale such as environmental toxin levels in a river. Furthermore, the ability to log the internal state of a single cell provides many opportunities for deeper understanding and research into how individual biological cells operate in both health and disease states within their normal, living context. The ability to create such logs is further enhanced by a mechanism that can track the timing of logged events.

SUMMARY

Internal and external states or stimuli (e.g., temperature, pH, levels of a given molecule, membrane-bound receptors for light, chemicals, or other stimuli or measurable quantity that may be transduced via proteins and/or ribonucleic acid (RNA)) of a cell may be recorded into a stable polynucleotide (e.g., deoxyribonucleic acid (DNA), RNA, or DNA-RNA hybrids) memory that resides within the cell. This record of the sensed states or stimuli may be heritable and passed to subsequent cellular generations. The record can be read by sequencing of the polynucleotide. Thus, the stable polynucleotide creates a record analogous to a log file of states or stimuli experienced by the cell. Particular nucleic acid sequences may also be incorporated into the stable polynucleotide in response to events that occur at known times. Use of the time-correlated inserts can provide temporal information together with the record of the sensed states or stimuli.

Precise gene editing techniques such as CRISPR/Cas (Clustered regularly interspaced short palindromic repeats/CRISPR associated protein) systems and TALEN (transcription activator-like effector nucleases) enable manipulation of polynucleotides in a way that incorporates pre-determined polynucleotide sequences into an existing polynucleotide. With these techniques, a record may be sequentially written to a polynucleotide so that the signal being recorded does not require any external control signals and may be performed through automatic, periodic sampling of an input state. The cell may be modified by addition of a vector that includes genes or an operon which creates gene products used for logging molecular events. Timing of events may be recorded by manually exposing the cell to a condition that results in incorporation of a particular sequence that can later be correlated with the time of exposure. Use of a gene oscillator or other cellular machinery that creates a periodic signal can also be used to trigger the insertion of polynucleotide sequences that record the passage of time.

A polynucleotide in the cell may be cut to create a double strand break (“DSB”). A new polynucleotide sequence may be inserted into the DSB using homology directed repair (“HDR”). Making the DSB and integrating the HDR templates in response to signals creates a record in the polynucleotide of events experienced by the cell and their timing.

DETAILED DESCRIPTION

This disclosure presents techniques for recording molecular events sensed by a cell and timing associated with those events in the genetic material of that cell. The molecular events and the timing events, referred to as “clock signals,” are both integrated into genetic material of the cell creating a stable, heritable record. The cell can be modified to incorporate known polynucleotide sequences when exposed to particular signals. This provides a log of the molecular events sensed by the cell. The cell can also be modified to incorporate known polynucleotide sequences at specific time points either manually triggered or in response to a natural or synthetic cellular cycle. Both molecular events and timing may be recorded together to provide a log that indicates the time specific molecular events were experienced by the cell. Alternatively, either may be implemented separately to provide a log of molecular events without timing information or to track passage of time independent of specific molecular events.

The genetic material is a “polynucleotide” which is often DNA but may also be RNA or a hybrid combination of DNA and RNA. The polynucleotide may or may not include one or more artificial nucleotides (e.g isoguanine, isocytosine, diaminopurine, etc.). References to “DNA” herein are understood to include all types of polynucleotides unless context specifically indicates otherwise. The detectable, molecular event may be an intra-cellular or extra-cellular event that results in generation of a signal that can be detected by a cellular system. Examples of molecular events include exposure to a chemical, a change in temperature, exposure to light (or dark), exposure to radiation, the presence of an antigen, a change in ionic concentration such as pH, etc. The molecular event may represent an external environmental condition or internal condition experienced by the cell. Timing indicators are also included in the genetic material of the cell. Timing indicators may be generated by manual action such as periodically exposing the cell to a condition that results in integration of a known sequence of DNA into the genome. This timing can be recorded and then later identification of these in such sequences may be correlated with the recorded times. Alternatively, the cell may be modified to include a mechanism such as a genetic oscillator that periodically causes integration of DNA into the genome. The timing of the oscillations can be used to identify the relative difference in time between two different instances in which DNA was integrated into the genome.

The cellular system that detects the molecular event may include an extra-cellular receptor that responds to conditions outside the cell or an intra-cellular receptor that responds to conditions within the cell. The cellular system may communicate a signal detected by a receptor through a signaling pathway that ultimately results in modification of a polynucleotide. The signaling pathway may be an “engineered signaling pathway” that is a natural signaling pathway modified in part or an entirely synthetic pathway that is added to the cell. A signaling pathway may cause changes in a polynucleotide by controlling the expression of a gene product. Expression may be controlled by interaction between the signaling pathway and an inducible promoter. A promoter is a region of DNA that initiates transcription of a particular gene. In response to the signal, the signaling pathway may either turn on an inducible promoter, which increases transcription of the associated gene, or repress a promoter, which decreases transcription of the associated gene. The gene product from a gene controlled by the promoter is the component that is used to modify the polynucleotide. For example, the gene product may be an enzyme that cuts the polynucleotide or the gene product may be another polynucleotide that is used to modify the polynucleotide that already exists in the cell.

Modification of the polynucleotide in the cell may be performed through homology directed repair (“HDR”). HDR uses a template polynucleotide (usually DNA but RNA may also be used) to repair a double-stranded break (“DSB”) in the polynucleotide. The repair removes the DSB and, based on the design of the template polynucleotide, referred to as an “HDR template” in this disclosure, may also add an additional polynucleotide sequence at the point of repair. Thus, a signal may cause a DSB to be created and then repaired through HDR in a way that adds a particular, additional polynucleotide sequence into the genetic material of the cell. Thereafter, presence of this polynucleotide sequence in the cell is an indication that the cell has experienced a particular molecular event. Design of engineered signaling pathways and homology repair templates creates an arbitrary association between a particular signal and a particular polynucleotide sequence. Similarly, HDR may be used to add polynucleotide sequences that represent particular time points. Thus, the molecular mechanism for integrating a new sequence into a polynucleotide is the same, but the significance is different depending on whether it was triggered by a particular molecular event or by something that occurred with a known timing.

By way of example only, a bacterial cell may be modified with a receptor that detects a particular chemical. The signal generated by that receptor may be passed through a signaling pathway to a promoter that increases the transcription of an HDR template that adds a particular DNA sequence, e.g. ACTAGA, to the genomic DNA of the bacterial cell when repairing a DSB. An enzyme creates a DSB at a predetermined position in the genomic DNA of the bacterial cell. The particular location of this DSB is specific based on the properties of the enzyme and is designed to be repaired by the corresponding HDR template. The chemical is detected by the receptor, which in turn leads to increased transcription of the homology repair template. When many copies of the homology repair template are present, one of those copies may be used to repair the DSB and add the sequence ACTAGA to the genomic DNA of the bacterial cell. Addition of the HDR template in response to detection of the chemical adds a new DNA sequence which itself may include a cut site for a different enzyme (e.g., there may be a cut site in the middle of the ACTAGA sequence). This other enzyme can create a DSB in the DNA added by the HDR template into which a second HDR template is incorporated that correlates with a time. The second HDR template may add, for example, the sequence GCT at the location of the cut site. This bacterial cell may be designed so that there are no cut sites for this other enzyme until after detection of the chemical and incorporation of the first HDR template. Thus, this construction adds a time indicator with each log of exposure to the chemical. Later, analysis of the DNA of the bacterial cell by DNA sequencing can detect the sequence ACT-GCT-AGA which then serves as a record that the cell was exposed to this particular chemical and indicates that the cell was exposed at the time that the second HDR template was available.

Homology Directed Repair

HDR is a mechanism in cells to repair DSBs. The most common form of HDR is homologous recombination. The HDR repair mechanism can be used by the cell when there is a homologous piece of DNA present to repair the DSB. HDR is considered a highly accurate mechanism for DSB repair due to the requirement of sequence homology between the damaged and intact donor strands of DNA. The process is nearly error-free if the DNA template used for repair is identical to the original DNA sequence at the DSB, or it can introduce very specific mutations into the damaged DNA if there are differences between the DNA template use for repair and the original DNA sequence. This disclosure discusses use of a homology repair template that adds a new DNA sequence at the point of the DSB as part of the repair process.

HDR includes homologous recombination (HR) and single-strand annealing (SSA) (Lieber. 2010 Annu. Rev. Biochem. 79:181-211). The most common form of HDR is HR which has the longest sequence homology requirements between the donor and acceptor DNA. Other forms of HDR include single-stranded annealing (SSA) and breakage-induced replication, and these require shorter sequence homology relative to HR. HDR at nicks (single-stranded breaks) can occur via a mechanism distinct from HDR at DSBs (Davis and Maizels. PNAS (0027-8424), 111 (10), p. E924-E932).

The terms “homology” and “homologous” as used herein in reference to nucleotide sequences refer to a degree of complementarity with other nucleotide sequences. There may be partial homology or complete homology (i.e., identity). A nucleotide sequence which is partially complementary, i.e., “substantially homologous,” to a nucleic acid sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid sequence. The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence to a target sequence under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.

The terms “homology” and “homologous” as used herein in reference to amino acid sequences refer to the degree of identity of the primary structure between two amino acid sequences. Such a degree of identity may be directed a portion of each amino acid sequence, or to the entire length of the amino acid sequence. Two or more amino acid sequences that are “substantially homologous” may have at least 50% identity, preferably at least 75% identity, more preferably at least 85% identity, most preferably at least 95%, or 100% identity.

By “hybridizable” or “complementary” or “substantially complementary” it is meant that a polynucleotide (e.g. DNA or RNA) comprises a sequence of nucleotides that enables it to non-covalently bind, to another polynucleotide in a sequence-specific, antiparallel, manner (i.e., a polynucleotide specifically binds to a complementary polynucleotide) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength. As is known in the art,

Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein; and Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). The conditions of temperature and ionic strength determine the “stringency” of the hybridization.

It is understood in the art that the sequence of polynucleotide need not be 100% complementary to that of its target polynucleotide to be specifically hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). A polynucleotide can comprise at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence complementarity to a target region within the target polynucleotide sequence to which they are targeted. For example, an antisense polynucleotide in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining non-complementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. Percent complementarity between particular stretches of polynucleotide sequences within polynucleotides can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

FIG. 1shows an illustrative schematic of operations to add a new DNA sequence into a double-stranded DNA (dsDNA)100through HDR. The new DNA sequence may become the record of a molecular event experienced by a cell containing the dsDNA100. The dsDNA100includes a target site102that directs an enzyme104to create a DSB in the dsDNA100within the target site102at a specific cut site106. The DSB may be created with blunt ends or with sticky ends depending on the specific enzyme and technique for making the DSB. The target site102is a sequence of DNA recognized by an enzyme that creates DSBs in dsDNA. By “enzyme reactive conditions” it is meant that any necessary conditions are available in an environment (i.e., such factors as temperature, pH, and lack of inhibiting substances) which will permit the enzyme to function. Enzyme reactive conditions can be either in vitro, such as in a test tube, or in vivo, such as within a cell.

The target site102may be intentionally introduced into the dsDNA100to enable the manipulations described below. Alternatively, a pre-existing portion of the dsDNA100may be selected as the target site102. If a pre-existing portion of the dsDNA100is selected as the target site102, then the sequence of other components of the system will be designed with reference to the sequence of the target site102. In some implementations, the target site102is unique such that there is only one target site102in the entire dsDNA strand and/or only one target site102throughout all the DNA in the cell. The dsDNA100may be genomic DNA inside a living prokaryotic or eukaryotic cell, DNA introduced to a living cell such as a plasmid or vector, or DNA in a cell-free system. The dsDNA100may exist as either linear or circular DNA prior to introduction of the DSB.

The enzyme104that creates the DSB may be any protein, protein-RNA complex, or protein-DNA complex (including multimeric complexes) that has the property of creating a DSB in dsDNA at the cut site106. Non-limiting examples of suitable enzymes include restriction enzymes, homing endonucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR/Cas, and NgAgo. These types of enzymes are all examples of site-specific nucleases that are capable of causing a DSB at a cut site106within a target site102. Further details about site-specific nucleases are provided below.

After creating a DSB at the cut site106, the target site102is split into two subsequences102(A),102(B) on either side of the DSB. Each of the two subsequences102(A),102(B) may, in an implementation, be between 5 and 20 nucleotides (nt) in length. Thus, the target site102may, in an implementation, be between 10 and 40 nt in length. In some implementations, the two subsequences102(A),102(B) may contain identical DNA sequences. The cut site106may be located in the middle of the target site102or it may be located elsewhere within the target site102. The schematic shown inFIG. 1illustrates a DSB with blunt ends, but as described above DSBs with sticky ends are also covered within the scope of this disclosure.

AN HDR template108is brought into proximity of the dsDNA100with the DSB. The HDR template108is single strand (ss) DNA or ssRNA. The HDR template repairs the DSB and inserts a polynucleotide sequence through the process of homology directed repair. HDR templates used to create specific mutations or insert new elements into a gene require a certain amount of homology surrounding the target site that will be modified. Thus, the HDR template108includes a 3′-end sequence110complementary to the first subsequence of the target site102(A) and a 5′-end sequence112complementary to a second subsequence of the target site102(B). Because they are complementary sequences, the length of the 3-end sequence110and the 5′-end sequence112are the same or about the same as the respective subsequences of the target site102(A),102(B). Thus, both 3-end sequence110and the 5′-end sequence112may be between 5 and 20 nt in length. The middle portion of the HDR template108contains a region114encoding a second target site116. This middle region114may contain two subsequences114(A),114(B) on either side of the point where the second target site116will be cut by a second enzyme. The length of the two subsequences114(A),114(B) in the middle portion114of the HDR template108may be different than the lengths of the two subsequences102(A),102(B) but may follow the same size range and be between five and 20 nt in length. Thus, the total length of the HDR template108may be between about 20 and 80 nt. Because the middle region114encodes a second target site116, the HDR template108itself provides the basis for this process to be repeated iteratively. So long as a signal is detected by a cell and the components for creating a DSB and performing HDR are available, this process may continue until the signal ceases. Thus, a length of the inserted DNA may correlate with a duration of the signal.

The HDR template108then repairs the DSB through HDR. The efficiency of HDR may be low, and in some conditions, other repair mechanisms can predominate. The efficiency of HDR is determined in part by the concentration of donor DNA present at the time of repair, the length of the homology arms of the donor DNA, the cell cycle, and the activity of the endogenous repair systems. An overabundance of the HDR template108may be provided to increase efficiency of HDR. The overabundance of the HDR template108may be provided to a cell-free system by adding additional copies of the ssRNA or ssDNA manually or with the use of microfluidics. The HDR template108may also be provided, in overabundance if desired, by placing a gene encoding the HDR template108under control of a strong promoter and/or by having multiple copies of the gene encoding the HDR template108all undergoing transcription. In an implementation, this promoter may be regulated by a signaling pathway that responds to a signal. When the signal is detected, the promoter is turned on and more copies of the HDR template108are generated.

The 5′-ended DNA strand is resected at the DSB to create a 3′ overhang. This will serve as both a substrate for proteins required for strand invasion and a primer for DNA repair synthesis. The HDR template108can then displace one strand of the homologous DNA duplex and pair with the other; this causes formation of hybrid DNA referred to as the displacement loop (“D loop”)118. The recombination intermediates can then be resolved to complete the DNA repair process. As mentioned above, an overabundance of the HDR template108may be provided. One of ordinary skill in the art will understand how to perform HDR with dsDNA100having a DSB and an HDR template108. Possible protocols for performing HDR are provided in Jie Liu et al.,In Vitro Assays for DNA Pairing in Recombination-Associated DNA Synthesis,745 Methods Mol. Bio. 363 (2011); Gratz, S. et al.,Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila,196 Genetics 967 (2014); Richardson, C. C. et al.,Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9using asymmetric donor DNA,34 Nature Biotechnology 399 (2016); and Lin, S. et al.,Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9delivery, eLIFE (2014).

After the HDR template108invades the dsDNA, the D loop118is formed by hybridization of the 3′-end sequence110to the first subsequence102(A) of the target site102and hybridization of the 5′-end sequence112to the second subsequence102(B) of the target site102. DNA polymerase synthesizes new ssDNA120complementary to the middle portion114of one strand of the dsDNA100. DNA ligase joins the sugar-phosphate backbone of the newly synthesized ssDNA120with the remainder of that strand of the dsDNA100. This forms one strand of the second target site116.

Hybridization requires that the two polynucleotides contain complementary sequences, although mismatches between bases are possible. The conditions appropriate for hybridization between two polynucleotides depend on the length of the polynucleotides and the degree of complementation which are variables well known in the art. The greater the degree of complementation between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of polynucleotides having those sequences. For hybridizations between polynucleotides with short stretches of complementarity (e.g. complementarity over 35 nt or less, 30 nt or less, 25 nt or less, 22 nt or less, 20 nt or less, or 18 nt or less) the position of mismatches becomes important. This is understood by one of ordinary skill in the art and described in Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001) at sec. 11.7-11.8. Typically, the length for a hybridizable polynucleotide is at least about 10 nt. Illustrative minimum lengths for a hybridizable polynucleotide are: at least about 15 nt; at least about 20 nt; at least about 22 nt; at least about 25 nt; and at least about 30 nt). Furthermore, the skilled artisan will recognize that the temperature, pH, and wash solution salt concentration may be adjusted as necessary according to factors such as length of the region of complementation and the degree of complementation.

Following repair of the first strand of the dsDNA100, the second strand of the dsDNA100is repaired by DNA polymerase and DNA ligase using the sequence of the new ssDNA120in the repaired, first strand as a template. This completes the repair of the dsDNA100resulting in dsDNA that includes the second target site116inserted within the first target site102.

DNA polymerases are enzymes that synthesize DNA molecules from individual deoxyribonucleotides. During this process, DNA polymerase “reads” an existing DNA strand to create a new, complementary strand. DNA ligase is a specific type of enzyme, a ligase, that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. It plays a role in repairing single-strand breaks. The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3′ hydroxyl ends of one nucleotide, (“acceptor”) with the 5′ phosphate end of another (“donor”). The DNA ligase from bacteriophage T4 is the ligase most-commonly used in laboratory research. It can ligate cohesive or “sticky” ends of DNA, oligonucleotides, as well as RNA and RNA-DNA hybrids, but not single-stranded polynucleotides. It can also ligate blunt-ended DNA.

Note that the HDR template108includes two types of regions: end regions and a middle region. The end regions are homologous to one of the strands of the dsDNA100on either side of the DSB. Here, the homologous regions are shown by the 3-end sequence110and the 5′-end sequence112. The homology need not be 100% but only to the extent that the 3′-end sequence110and the 5′-end sequence112hybridize to one strand of the dsDNA100. The middle region is the middle portion114of the HDR template108that encodes the sequence of the second target site116. Independently varying both the end regions and the middle region allows for creation of multiple different HDR templates108from a relatively limited set of end regions and middle regions. Thus, the middle region of an inserted HDR template108need not have the same target site102or cut site106as the dsDNA100it is being inserted into.

Following HDR, the dsDNA100includes the first subsequence102(A) of the first target site102followed by the first subsequence116(A) of the second target site116. The DNA sequence122represented by this order of the two subsequences102(A),116(A) of the two target sites may represent a particular signal combination (e.g., temperature above 30° C. followed by pH under 5). As mentioned above, a length of the subsequence102(A) is from five to 20 nt and the length of the subsequence114(A) is also from five to 20 nt. Thus, in an implementation, the total length of the DNA sequence122is from 10 to 40 nt.

HDR, however, is not the only way to repair a DSB. Non-Homologous End-Joining (NHEJ) is a pathway that repairs double-strand breaks in DNA and may be favored over HDR in many conditions. NHEJ is referred to as “non-homologous” because the break ends are directly ligated without the need for a homologous template. NHEJ is active throughout the cell cycle and has a higher capacity for repair, as there is no requirement for a repair template (sister chromatid or homologue) or extensive DNA synthesis. NHEJ also finishes repair of most types of breaks in tens of minutes—an order of magnitude faster than HDR. Thus, in many cells there is competition between HDR and NHEJ. If the ratio of HDR to NHEJ is high enough, HDR will continue. However, in the presence of NHEJ some of the DSBs formed by the enzyme104will rejoin without an insert.

NHEJ is consequently the principle means by which DSBs are repaired in natural cells. NHEJ-mediated repair is prone to generating indel errors. Indel errors generated in the course of repair by NHEJ are typically small (1-10 nt) but extremely heterogeneous. There is consequently about a two-thirds chance of causing a frameshift mutation. Thus, it may be desirable to minimize NHEJ and increase the probability that a DSB will be repaired by HDR. The likelihood of HDR being used may be improved by inhibiting components of the NHEJ process. Addition of small molecules such as NU7441 and KU-0060648 is one technique for inhibiting NHEJ through inhibition of DNA-dependent protein kinase, catalytic subunit (“DNA-PKcs”). Techniques for enhancing HDR efficiency in this way are described in Maruyama, et al.,Increasing the efficiency of precise genome editing with CRISPR-Cas9by inhibition of nonhomologous end joining.33(5) Nature Biotechnology, 538 (2015) and Robert, et. al.,Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing.7 Genome Medicine 93 (2015). In an implementation, HDR efficiency may be improved by suppressing the molecules KU70, KU80, and/or DNA ligase IV, which are involved in the NHEJ pathway. In addition to the suppression, the Cas9 system, EB55K, and/or E4orf6 may be expressed to further increase HDR efficiency and reduce NHEJ activity. Techniques for enhancing HDR efficiency in this way are described in Chu et al.,Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells.33(5) Nature Biotechnology, 543 (2015). Further, use of a single-stranded DNA oligo donor (ssODN) has been shown to improve the rate of HDR and knockin efficiency by up to 60% in Richardson et al.,Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9using asymmetric donor DNA,34(3) Nature Biotechnology 339 (2016).

FIG. 2shows schematic illustrations of further manipulations performed on the dsDNA100molecule ofFIG. 1. A second enzyme200creates a second DSB at a second cut site202in the second target site116. The second target site116has a different sequence than the first target site102, and thus, the second enzyme200recognizes a different DNA sequence than the first enzyme104. Creating a DSB in the second target site116at the cut site202creates the first subsequence116(A) of the second target site116on one side of the cut site202and a second subsequence116(B) of the second target site116on the other side of the cut site202. In some implementations, the first subsequence116(A) and the second subsequence116(B) may have the same sequence. Thus, the first subsequence116(A) and a second subsequence116(B) may have the same nucleotide length. Also, if the first subsequence116(A) and the second subsequence116(B) are the same sequence, the second target site116may be thought of as having a single subsequence repeated once with a cut site202in the middle.

A second HDR template204contacts the dsDNA100to provide a template for HDR of the DSB. The second HDR template204includes a 3′-end region206that is homologous to one strand of the dsDNA100within the first subsequence116(A) of the second target site116. The second HDR template204also include a 5′-end region208that is homologous to one strand of the dsDNA100within the second subsequence116(B) of the second target site116. The second HDR template204also includes a portion in the middle210that encodes a third target site for a third enzyme. The middle region210includes a first subsequence210(A) on one side of a third cut site212and a second subsequence210(B) on other side of the third cut site212.

Annealing of the second HDR template204to one strand of the dsDNA100creates a D loop214by hybridization of the 3′-end sequence206to the subsequence116(A) and hybridization of the 5′-end sequence208to the subsequence116(B). DNA polymerase and DNA ligase repair the strand of the dsDNA100to which the second HDR template204is hybridized by creating new DNA216. The second strand of the dsDNA100is then repaired using the first strand as a template.

The dsDNA100now includes the third target site218inserted into the middle of the second target site116(which is itself inserted in the middle of the first target site102). The order of the subsequence116(A) followed by the subsequence218(A) may create a record of a second combination of detected signals. Thus, the growing string of inserted DNA sequences can provide an ordered log of molecular events experienced by a cell. This process can repeat to record any number of molecular events.

Addition of HDR templates into existing DNA using the mechanisms described above may be regulated by signaling pathways as described in detail below. The encoding scheme described herein allows for insertion of DNA sequences representing an unbounded length. AN HDR template that does not include a cut site may be added once, end the process of HDR, and create a record that a specified signal was detected. The dsDNA in a cell may have multiple different target sites at different locations that include different cut sites and are homologous to different HDR templates. This provides for orthogonal recording of signals without any linkage between the signals. For example, a first target site may be configured to integrate a first HDR template if the cell is exposed to radiation, a second target site may be configured to integrate a second HDR template if the cell is exposed to hydrocarbons, and a third target site may be configured to integrate a third HDR template if the cell is exposed to light. Each cell configured in this way will create independent logs of the signals (e.g., radiation, hydrocarbons, and light) that it was exposed to. A cell may be modified to have any number of orthogonal target sites.

The three target sites may be represented as X1X2, Y1Y2, and Z1Z2. The first portion of the target site (e.g, X1, Y1, or Z1) corresponds to subsequence102(A) or subsequence116(A) shown inFIG. 1. The remaining portion of the target site (e.g., X2, Y2, or Z2) corresponds to subsequence102(B) or subsequence116(B) shown inFIG. 1. Thus, each X, Y, and Z represents a DNA sequence of about 5 to 20 nt such as, for example only, ACTGAA, GCCTCAT, TGACG, etc. In some implementations X1=X2, etc., but in other implementations the first portion of a target site may be different in sequence and/or length from the remaining portion of the target site.

The HDR templates all have end regions that are homologous to one of the target sites. Thus, the HDR templates will have sequences of the structure: X1aX2, Y1bY2, and Z1cZ2where “a,” “b,” and “c” represent DNA sequences of the middle regions. Recall that the middle region of the HDR templates may itself encode a target site. Thus, for example, a may represent X1X2, b may represent Z1Z2, and c may represent a different target site W1W2. If the middle region does encode a target site, integration of an HDR template into dsDNA may be followed by further integration of the same or a different HDR template. Insertion of an HDR template into dsDNA that has been itself created by integration of an HDR template is referred to in this disclosure as “iterative integration.”

Thus, a design using iterative integration of a single HDR template may record the presence of a signal and the length of the signal. For example, the HDR template may be XaXXaX and the initial insertion site may be XX. Iterative integration will result in a sequence that is represented by:XXaXaXaXaX . . . XaXaXaXaXX
This sequence can keep growing continuously while the signal is detected. A potential problem is that the HDR templates may be cut by the same enzyme that creates a DSB at the insertion site because both include the sequence XX which is recognized by the enzyme used for this logging. Physical separation, splicing, self-excising elements, homologous bridges, or methylation may be used to prevent or decrease the amount of HDR templates that are cut before integration into the dsDNA.

In one configuration, the continued detection of multiple signals may be recorded by appropriately designed HDR templates and insertion sites. AN HDR template with a sequence XaYYaX is expressed when a first signal “a” is detected. Similarly, an HDR template YbXXbY is expressed when a second signal “b” is detected. Initially, the cell may include a target site XX or YY. If the cell only includes the target site XX, presence of signal “b” will not be recorded until the HDR template associated with signal “a” is first integrated into the DNA of the cell. As each HDR template provides the target site for the other, alternating exposure to signals “a” and “b” or continued exposure to both signals leads to continued integration of the HDR templates. This alternating, iterative addition will result in a sequence represented by:XaYbXaYbX . . . XbYaXbYaX
This provides sequential recording of signals “a” and “b” independent of the relative concentrations of the HDR templates XaYYaX and YbXXbY. This technique for logging multiple signals at the same location in DNA may be expanded to cover three, four, or even more different signals.

In one configuration, multiple signals may be associated with HDR templates that have the same target sites. For example, a first signal “a” and a second signal “b” may be associated respectively with the HDR templates XaXXaX and XbXXbX. Either HDR template may be integrated into the target site XX. Once integrated, both HDR templates also include the target site XX allowing for iterative addition of either or both. In most conditions, the level of relative incorporation of the two HDR templates will be proportional to the relative concentrations of HDR templates. The amount of each HDR template present in the cell may be designed to be proportional to the strength, frequency, and/or duration of the corresponding signal. For example, if signal “a” is strong and constant the cell may produce a relatively large amount of the XaXXaX template. When signal “b” is present, the amount of the XbXXbX template may increase and then that HDR template is also integrated into the DNA of the cell. So long as all components are present, iterative insertion of these two templates depends on relative strengths of signals “a” and “b” and will result in a sequence represented by:X[a|b]X[a|b]X . . . X[a|b]X[a|b]X
where [a|b] is a or b. The relative amount of “a” vs. “b” in the DNA provides a record of which signal was strongest and changes from a period of “a” dominance to a period of “b” dominance indicates a temporal change in the relative signal strengths. This configuration may be expanded to include three, four, or more different signals and HDR templates. Analysis of the DNA sequence created by this iterative and competitive integration of multiple HDR templates may be performed over defined lengths of nucleotides which represent periods of time. The lengths of nucleotides may be analyzed by considering a series of sliding windows (e.g., a 10,000 nt stretch of the DNA) and determining the relative level of Xa vs. Xb in a given window. This provides information about the relative strength of signals “a” and “b” during a given period of time.

One way of using this configuration is in a cell that has constitutive expression (rather than in response to a signal) of the first HDR template XaXXaX. This template will be expressed and present in the cell at a constant level. It may be thought of as a background signal. The level of the second HDR template XbXXbX will vary depending on the strength of signal “b.” Thus, the amount of the XbXXbX template integrated into the DNA indicates the relative strength of signal “b” as compared to the baseline established by expression of XaXXaX.

Another way of using the configuration described above is to use the presence of one of the HDR templates in the DNA of the cell as a temporal indication like a time stamp. For example, the concentration of the first HDR template may respond to the detection of a signal. If the signal is continually present, then the HDR template XaXXaX will be iteratively introduced into the DNA of the cell. As described above, the length of the insertion will depend on the duration that the signal “a” is present. Intentionally exposing the cell to signal “b” at known time points provides references point in the DNA that can be correlated to the known times of exposure to signal “b.” When exposed to signal “b,” the expression of the second HDR template XbXXbX increases to a level greater than the expression of XaXXaX (e.g., the second HDR template may be regulated by a stronger promoter or present in more copies than the first HDR template). Thus, each point in the DNA that has an insertion of XbXbXb . . . indicates a time when the cell was exposed to “b” For example, if the cell is exposed to signal “b” every 24 hours, each string of DNA between XbXbXb . . . sequences represents the activity of signal “a” during that 24-hour period.

The above configurations may be combined to record multiple signals sequentially regardless of relative strength and also to record the strongest signal based on competing HDR templates. There may be multiple classes of HDR templates with each class having multiple different HDR templates transcribed in response to different signals. For example, there may be two classes of HDR templates XaYYaX and YbXXbY. Because these two HDR templates integrate into the target site created by addition of the other (i.e., the template that integrates into XX adds the target site YY and the template that integrates into YY adds the target site XX) they will alternate. Thus, the DNA will incorporate first an HDR template from the “a” class then an HDR template from the “b” class. Each class of HDR template includes two (but may include any number) HDR templates with partially different sequences that correspond to different signals. Thus, a signal “a1” may cause increased expression of the HDR template Xa1YYa1X and a signal “a2” may cause increased expression of the HDR template Xa2YYa2X. Similarly, a signal “b1” may cause increased expression of the HDR template Yb1XXb1Y and a signal “b2” may cause increased expression of the HDR template Yb2XXb2Y. If the cell begins with DNA that includes the insertion site XX, then first one of the “a” HDR templates will be integrated based on the relative concentrations of the Xa1YYa1X and of the Xa2YYa2X HDR templates. Doing so creates a YY insertion site and is followed by integrating one of the “b” HDR templates again based on relative concentrations.

In one implementation, each class of the HDR template may record values associated with a particular type of molecular event. For example, the “a” class of HDR templates may indicate temperature experienced by the cell with Xa1YYa1X expressed if the temperature is below 32° C. and Xa2YYa2X expressed if the temperature is above 42° C. Thus, integration of the “a” class of HDR templates creates a record of relative temperature. The “b” class of HDR templates may be associated with a different type of signal such as salinity. The HDR template Yb1XXb1Y may be expressed when the cell is in an environment with salinity below 0.600 M and Yb2XXb2Y may be expressed when the cell is in an environment with salinity above 0.700 M. Thus, the record created in the DNA of this cell shows temperature high/low and salinity high/low. Each is recorded in turn so there is a log created over time showing changes in two different signals. Of course, any number of different gradations or levels of variables may be tracked by having distinct HDR templates under the control of appropriate promoter.

In one example implementation, using Cas9 as the nuclease with a PAM sequence of NNNNGATTT as the enzyme, three target sites may be:

Each of the target sites is recognized by a corresponding guide ssDNA that cuts the dsDNA at the location indicated by the “{circumflex over ( )}” below. They should have a trans-activating crRNA (tracrRNA) that is a small trans-encoded RNA for attaching to Cas9 appended to the end. The crRNAs are incorporated into effector complexes, where the crRNA guides the complex to the target site and the Cas proteins create a DSB in the polynucleotide. The respective ssDNA sequences are:

(SEQ ID NO: 1)gX1= TAGCCGTATCGAGCATCGATG{circumflex over ( )}CGC(SEQ ID NO: 3)gY1= GATCGATGGACTCTGCATCTA{circumflex over ( )}TCG(SEQ ID NO: 5)gZ1= CGGGACGATCGATCGGGCTAG{circumflex over ( )}ACT
Then a homology directed repair sequence of X1Y1Y2X2is: TAGCCGTATCGAGCATCGATG|GATCGATGGACTCTGCATCTA|TCGNNNNGATT|CGCNNNNGATT (SEQ ID NO: 7) and a homology directed repair sequence of Y1X1X2Y2is: GATCGATGGACTCTGCATCTA|TAGCCGTATCGAGCATCGATG|CGCNNNNGATT|TCGNNNNGATT (SEQ ID NO: 8). Other homology directed repair sequences can be designed according to the same pattern.

An initial cut of the target site X1X2will create a DSB that appears as (only one strand of the dsDNA is shown):

After HDR with X1Y1Y2X2, one strand of the dsDNA will have the following sequence that now includes the target site Y1Y2indicated by italics:

(SEQ ID NO: 7)TAGCCGTATCGAGCATCGATG|GATCGATGGACTCTGCATCTA||TCGNNNNGATT|CGCNNNNGATT.
The dsDNA is now able to be cut by a Cas9 that has Y1creating a DSB at the location represented by “∥”. HDR may be performed with Y1X1X2Y2, for example, further adding to the dsDNA and completing another iteration of encoding. This may be continued with various sequences of cuts and HDR templates to record any series of molecular events.
Signaling Pathways

FIG. 3shows a diagram300of an illustrative signaling pathway that regulates expression of a gene. The signaling pathway may be an engineered signaling pathway that is created or modified in some way to be different from a wild-type signaling pathway. The signaling pathway controls the expression of a gene302that is under the control of a promoter304and may also be under the control of an operator306. A promoter is a region of DNA that initiates transcription of a particular gene. Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA (towards the 5′ region of the sense strand). Illustrative promoters are described below. The sequence of the promoter region controls the binding of the RNA polymerase and transcription factors. An operator is a segment of DNA to which a repressor binds to decrease or stop gene expression. A “transcription factor” is a protein that binds near the beginning of the coding sequence (transcription start site) for a gene or functional mRNA. Transcription factors are necessary for recruiting DNA polymerase to transcribe DNA. A transcription factor can function as a repressor, which can bind to the operator to prevent transcription. The gene302, the promoter304, and the operator306are on a dsDNA molecule that may be genomic DNA of a cell or other DNA such as a plasmid or vector. In some implementations, the promoter304may respond to signals such as temperature or pH and thus the promotor304itself may be the signaling pathway.

The repressor (and/or “knockdown”) may be a protein or mRNA (small hairpin loops (shRNA), interfering mRNA (RNAi or siRNA)) that binds to DNA/RNA and blocks either attachment of the promoter, blocks elongation of the polymerase during transcription, or blocks mRNA from translation. In addition to repressors, the CRISPR/Cas9 system itself may be used for sequence-specific repression of gene expression in prokaryotic and eukaryotic cells. Specifically, the technique of CRISPR interference (CRISPRi) uses catalytically dead Cas9 lacking endonuclease activity to regulate genes in an RNA-guided manner. Catalytically inactive Cas9 may be created by introducing point mutations into the Cas9 protein such as at the two catalytic residues (D10A and H840A) of the gene encoding Cas9. In doing so, dCas9 is unable to cleave dsDNA but retains the ability to target DNA. Targeting specificity for CRISPRi is determined by complementary base pairing of a guide RNA (gRNA) to the genomic loci. The gRNA may be designed to target a specific promoter. The complex catalytically dead Cas9 and the gRNA will block activation of the promoter and turn off expression of any gene regulated by that promoter.

The signaling pathway may include a signaling cascade308that carries a signal from a first messenger (i.e., the initial signal) and eventually results in activation, or alternatively suppression, of either the promoter304or the operator306. The initial signal that sets the signaling cascade308into action may be an internal or external signal. The signaling pathway may be a trans-membrane signaling pathway that includes an external receptor310which detects extracellular signals and communicates the signal across a membrane312. The membrane312may be a cell wall, lipid bilayer, artificial cell wall, or synthetic membrane.

In one implementation, the external receptor310may be a G protein-coupled receptor (GPCR). GPCRs constitute a large protein family of receptors, that sense molecules outside the membrane312and activate the signaling cascade308and, ultimately, cellular responses. The GPCR is activated by an external signal in the form of a ligand or other signal mediator. This creates a conformational change in the GPCR, causing activation of a G protein. Further effect depends on the type of G protein. G proteins are subsequently inactivated by GTPase activating proteins, known as RGS proteins. The ligands that bind and activate these GPCRs include light-sensitive compounds, odors, pheromones, hormones, neurotransmitters, etc. and vary in size from small molecules to peptides to large proteins. When a ligand binds to the GPCR it causes a conformational change in the GPCR, which allows it to act as a guanine nucleotide exchange factor (GEF). The GPCR can then activate an associated G protein by exchanging its bound GDP for a GTP. The G protein's a subunit, together with the bound GTP, can then dissociate from the β and γ subunits to further affect intracellular signaling proteins or target functional proteins directly depending on the a subunit type.

In one implementation, the external receptor310may be a photosensitive membrane protein. Photoreceptor proteins are light-sensitive proteins involved in the sensing and response to light in a variety of organisms. Photoreceptor proteins typically consist of a protein moiety and a non-protein photopigment that reacts to light via photoisomerization or photoreduction, thus initiating a change of the receptor protein that triggers the signaling cascade308. Pigments found in photoreceptors include retinal (retinylidene proteins, for example rhodopsin in animals), flavin (flavoproteins, for example cryptochrome in plants and animals) and bilin (biliproteins, for example phytochrome in plants). One example of engineered use of light-sensitive proteins is found in Tamsir, A. et al.,Robust Multicellular Computing Using Genetically Encoded NOR Gates and Chemical‘Wires’, 469 Nature 214 (2011).

The external receptor310, in some implementations, may also be a membrane-bound immunoglobulin (mlg). A membrane-bound immunoglobulin is the membrane-bound form of an antibody. Membrane-bound immunoglobulins are composed of surface-bound IgD or IgM antibodies and associated Ig-α and Ig-β heterodimers, which are capable of signal transduction through the signaling cascade308in response to activation by an antigen.

In one implementation, the external receptor310may be a Notch protein. The Notch protein spans the cell membrane, with part of it inside and part outside. Ligand proteins binding to the extracellular domain induce proteolytic cleavage and release of the intracellular domain, which enters the cell to modify gene expression. The receptor may be triggered via direct cell-to-cell contact, in which the transmembrane proteins of the cells in direct contact form the ligands that bind the notch receptor. Signals generated by the Notch protein may be carried to an operon by the Notch cascade which consists of Notch and Notch ligands as well as intracellular proteins transmitting the notch signal.

In one implementation, temperature may activate the signaling pathway. Thus, by altering the temperature, expression of the gene302may be up or down regulated. Temperature sensing molecules that occur naturally in single celled organisms include heat shock proteins and certain RNA regulatory molecules, such as riboswitches. Heat shock proteins are proteins that are involved in the cellular response to stress. One example of a heat shock protein that responds to temperature is the bacterial protein DnaK. Temperatures elevated above normal physiological range can cause DnaK expression to become up-regulated. DnaK and other heat shock proteins can be utilized for engineered pathways that respond to temperature. Riboswitches are a type of RNA molecule that can respond to temperature in order to regulate protein translation. An example of a temperature-regulated engineered pathway that has utilized a riboswitch can be found in Neupert, J. et al.,Design of simple synthetic RNA thermometers for temperature-controlled gene expression in Escherichia coli,36(19) Nucleic Acids Res., e124, (2008). Another example of a temperature-sensitive molecule that can be utilized to regulate engineered cell pathways is a temperature-sensitive mutant protein. Single mutations can be made to proteins, which cause the proteins to become unstable at high temperatures, yet remain functional at lower temperatures. Methods for synthesizing temperature-sensitive mutant proteins can be found in Ben-Aroya, S. et al.,Making Temperature-Sensitive Mutants,470 Methods Enzymology 181 (2010). An example of a temperature-controlled engineered pathway that utilizes a temperature-sensitive mutant can be found in Hussain, F. et al.,Engineered temperature compensation in a synthetic genetic clock,111(3) PNAS 972 (2014).

In one implementation, ion concentration or pH may activate the signaling pathway. With signaling pathways of this type, placing a cell in a different ionic environment or altering pH surrounding the cell may be used to control the availability of a given HDR template or enzyme. Examples of cellular sensing molecular mechanisms that detect ionic strength or pH include many viral proteins, such as herpes simplex virus gB, rubella virus envelope protein, influenza hemagglutinin, and vesicular stomatitis virus glycoprotein. An example of a natural cellular pathway that is regulated by pH is penicillin production byAspergillus nidulansas described in Espeso, E. et al.,pH Regulation is a Major Determinant in Expression of a Fungal Penicillin Biosynthetic Gene,12(10) EMBO J. 3947 (1993). Another example of a pH-sensitive molecule that can be utilized to regulate engineered cell pathways is a pH-sensitive mutant protein. Single mutations can be made to proteins, which can cause the proteins to become less stable in either acidic or basic conditions. For example, pH-sensitive antibodies can bind to an antigen at an optimal pH, but are unable to bind to an antigen at a non-optimal pH. A technique for creating pH-sensitive antibodies that can be used for engineered signaling pathways can be found in Schroter, C. et al.,A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display,7(1) MAbs 138 (2015). These and other similar sensing mechanisms may be engineered to affect the behavior of a promoter304or operator306.

The gene302encodes for gene product314that may ultimately be the basis for a number of components in an HDR system. For example, the gene product314may be translated into protein, used directly as RNA, or reverse transcribed into DNA. In one implementation, the gene product314may be translated into a nuclease316that creates DSBs such as, for example, enzyme104shown inFIG. 1, or enzyme200shown inFIG. 2. The nuclease316may be a Cas enzyme such as Cas9, Cas1, or Cas2.

For example, theS. pyogenesCas9 system from the Clustered Regularly-Interspaced Short Palindromic Repeats-associated (CRISPR-Cas) family is an effective genome engineering enzyme that catalyzes double-stranded breaks and generates mutations at DNA loci targeted by a gRNA. The native gRNA is comprised of a 20 nucleotide (nt) Specificity Determining Sequence (SDS), which specifies the DNA sequence to be targeted, and is immediately followed by a 80 nt scaffold sequence, which associates the gRNA with Cas9. In addition to sequence homology with the SDS, targeted DNA sequences possess a Protospacer Adjacent Motif (PAM) (5′-NGG-3′) immediately adjacent to their 3′-end in order to be bound by the Cas9-sgRNA complex and cleaved. When a double-stranded break is introduced in the target DNA locus in the genome, the break is repaired by either homologous recombination (when a repair template is provided) or error-prone non-homologous end joining (NHEJ) DNA repair mechanisms, resulting in mutagenesis of targeted locus. Even though the normal DNA locus encoding the gRNA sequence is perfectly homologous to the gRNA, it is not targeted by the standard Cas9-gRNA complex because it does not contain a PAM.

In a wild-type CRISPR/Cas system, gRNA is encoded genomically or episomally (e.g., on a plasmid). Following transcription, the gRNA forms a complex with Cas9 endonuclease. This complex is then “guided” by the specificity determining sequence (SDS) of the gRNA to a DNA target sequence, typically located in the genome of a cell. For Cas9 to successfully bind to the DNA target sequence, a region of the target sequence must be complementary to the SDS of the gRNA sequence and must be immediately followed by the correct protospacer adjacent motif (PAM) sequence (e.g. “NGG”). Thus, in a wild-type CRISPR/Cas9 system, the PAM sequence is present in the DNA target sequence but not in the gRNA sequence (or in the sequence encoding the gRNA).

The PAM sequence is typically a sequence of nucleotides located adjacent to (e.g., within 10, 9, 8, 7, 6, 5, 4, 3, 3, or 1 nucleotide(s) of) an SDS sequence). A PAM sequence is “immediately adjacent to” an SDS sequence if the PAM sequence is contiguous with the SDS sequence (that is, if there are no nucleotides located between the PAM sequence and the SDS sequence). In some implementations, a PAM sequence is a wild-type PAM sequence. Examples of PAM sequences include, without limitation, NGG, NGR, NNGRR(T/N), NNNNGATT, NNAGAAW, NGGAG, and NAAAAC, AWG, CC. In some implementations, a PAM sequence is obtained fromStreptococcus pyogenes(e.g., NGG or NGR). In some implementations, a PAM sequence is obtained fromStaphylococcus aureus(e.g., NNGRR(T/N)). In some implementations, a PAM sequence is obtained fromNeisseria meningitidis(e.g., NNNNGATT). In some implementations, a PAM sequence is obtained fromStreptococcus thermophilus(e.g., NNAGAAW or NGGAG). In some implementations, a PAM sequence is obtained fromTreponema denticolaNGGAG (e.g., NAAAAC). In some implementations, a PAM sequence is obtained fromEscherichia coli(e.g., AWG). In some implementations, a PAM sequence is obtained fromPseudomonas auruginosa(e.g., CC). Other PAM sequences are contemplated. A PAM sequence is typically located downstream (i.e., 3′) from the SDS, although in some embodiments a PAM sequence may be located upstream (i.e., 5′) from the SDS.

In one implementation, the gene product314encodes for gRNA318that is used by the Cas enzyme316to target a specific DNA sequence. The system may be designed to have all components needed for performing HDR other than the gRNA318. Thus, transcription of the gRNA in response to a signal provides the last component needed to perform HDR and results in incorporation of an HDR template thereby creating a log of the molecular event. Alternatively, the gRNA318may be used not to cut dsDNA but to turn off a promoter through use of CRISPRi guide RNA. CRISPRi guide RNA directs the Cas enzyme316to bind to the promoter304and prevent transcription of the gene302. In this design, the presence of a signal would stop the insertion of a particular HDR template.

A gRNA is a component of the CRISPR/Cas system. A “gRNA” (guide ribonucleic acid) herein refers to a fusion of a CRISPR-targeting RNA (crRNA) and a trans-activation crRNA (tracrRNA), providing both targeting specificity and scaffolding/binding ability for Cas9 nuclease. A “crRNA” is a bacterial RNA that confers target specificity and requires tracrRNA to bind to Cas9. A “tracrRNA” is a bacterial RNA that links the crRNA to the Cas9 nuclease and typically can bind any crRNA. The sequence specificity of a Cas DNA-binding protein is determined by gRNAs, which have nucleotide base-pairing complementarity to target DNA sequences. Thus, Cas proteins are “guided” by gRNAs to target DNA sequences. The nucleotide base-pairing complementarity of gRNAs enables, in some embodiments, simple and flexible programming of Cas binding. Nucleotide base-pair complementarity refers to distinct interactions between adenine and thymine (DNA) or uracil (RNA), and between guanine and cytosine. In some embodiments, a gRNA is referred to as a stgRNA. A “stgRNA” is a gRNA that complexes with Cas9 and guides the stgRNA/Cas9 complex to the template DNA from which the stgRNA was transcribed.

The length of a gRNA may vary. In some embodiments, a gRNA has a length of 20 nucleotides to 200 nucleotides, or more. For example, a gRNA may have a length of 20 to 175, 20 to 150, 20 to 100, 20 to 95, 20 to 90, 20 to 85, 20 to 80, 20 to 75, 20 to 70, 20 to 65, 20 to 60, 20 to 55, 20 to 50, 20 to 45, 20 to 40, 20 to 35, or 20 to 30 nt.

In one implementation, the gene product314may itself be or may encode for an HDR template320. The HDR template320may be, for example, the HDR template108shownFIG. 1or the HDR template204shown inFIG. 2. The gene product314, although it is a ssRNA, may be capable of functioning as an HDR template320due to the ability of RNA to hybridize with DNA. RNA transcript-mediated HDR has been shown to function successfully in eukaryotic cells. See Keskin, H. et al.,Transcript-RNA-templated DNA recombination and repair,515 Nature 436 (2014) and Storici. F. et al.,RNA-templated DNA repair,447 Nature 338 (2007). If RNA is used as the HDR template, the cell may be further modified to reduce or remove enzymes that degrade RNA-DNA hybrids. In one implementation, the cell using RNA as the HDR template may beS. cerevisiae. Additionally, complementary DNA (cDNA), resulting from reverse-transcription of mRNA, and/or transcript RNA itself may aid DSB repair via HDR. Moreover, splicing of both expressed RNA and potentially of mRNA can change the sequence of RNA that serves as a template for reverse transcriptase to synthesize cDNA. Thus, the cDNA used as an HDR template may have a different sequence, due to splicing, than genomic or other DNA encoding the initial RNA transcript. The gene product314may also be converted to ssDNA by reverse transcriptase and used as the HDR template320in the form of DNA.

The gene product314may also be translated into some other enzyme product322. The other enzyme product322represents another enzyme that may be used for logging of molecular events through HDR. Both DNA Taq polymerase and DNA ligase are examples of other enzyme products used for performing HDR. In a system that lacks one or both of these enzymes, regulated addition through control of gene expression is a way to regulate the ability to perform HDR. Other enzymes such as transcription factors are another type of other enzyme products322. Transcription factors expressed from a first gene may be used to activate the promoter or operator of a second gene. There may be greater need for addition of other enzyme products322in a cell-free system or in a minimal cell than in a biological cell that includes wild-type cellular machinery.

FIG. 4shows a diagram400of two illustrative signaling pathways that create different gene products at levels responsive to strengths of the respective signals. A first signaling pathway402responds to a first signal404by increasing activity of a first promoter406which controls transcription of a first gene408. The first signaling pathway402and the first signal404may be any of the signaling pathways or types of signals discussed in this disclosure. The first gene408creates a first gene product410that may be any of the types of gene products shown inFIG. 3. For purposes of illustration, the first gene product410is shown as encoding a first homology repair template412. Thus, an increase in the first signal404leads to an increase in the synthesis of the first homology repair template412.

Similarly, a second signaling pathway414is responsive to a second signal416by increasing activity of a second promoter418which controls transcription of a second gene420. The second gene420encodes a second gene product422. The second gene product422may be any of the types of gene products discussed inFIG. 3. The second gene product422may be the same or a different type of gene product than the first gene product410. In this diagram400, the second gene product422is shown as a second homology repair template424. The amount of the second homology repair template424is thus regulated by the strength of the second signal416.

If, for example, the second signal416is stronger and/or more frequent than the first signal404, the cell will create a greater number of copies of the second homology repair template424than of the first homology repair template412. The respective signaling pathways402,414and the promoters406,418may be selected to maintain a similar ratio of correspondence between respective signal strengths and synthesis of homology repair templates412,424. For example, the respective signaling pathways402,414may be the same except for the portion of the signaling pathway directly involved in sensing the primary signal. The promoters406,418may also be similar and different only in one aspect such as the specific transcription factor used to activate the promoter.

In this example, the second homology repair template424is present at a concentration that is twice as much as the first homology repair template412. This indicates that the second signal416is approximately twice as strong as the first signal404. Because the concentration of the second HDR template424is twice that of the first HDR template412, for each HDR event it is twice as likely that the second homology repair template424will be integrated into a section of dsDNA426. Thus, over a prolonged period of iterative integration of homology repair templates, it is likely that a sequence428from the second homology repair template424will be twice as common as a sequence430from the first homology repair template412. The dsDNA426may include, for example, a target site432into which either the first homology repair template412or the second homology repair template424may be inserted. The relative amount of integration of the sequence428from the second homology repair template424and the sequence430of the first homology repair template412into the dsDNA426reflects the relative concentrations of the first homology repair template412and the second homology repair template424. Specifically, in this example, the sequence428of the second homology repair template424is present twice as often as the sequence430from the first homology repair template412. Thus, the first HDR template412and the second HDR template424integrate into the dsDNA426in proportion to their respective concentrations.

If the strength of one or more of signals404,416in this example system changes over time then the relative concentrations of the corresponding HDR templates412,424will also change. This change over time may be observed by analyzing the sequence of the dsDNA426and observing throughout different portions of that sequence how the ratio of the sequence428of the second homology repair template424to the sequence430of the first homology repair template412varies. This temporal analysis may be implemented, for example, by analyzing a sliding window of nucleotides of the dsDNA426and counting the number of times the sequence428from the second homology repair template424is found and the number of times the sequence430of the first homology repair template412is found. The sliding window may be any length such as, for example 500 nt, 1000 nt, 5000 nt, etc.

FIG. 5shows an illustrative cell500that is capable of heritability storing a log of events experienced by the cell500. The cell500may be anE. colicell, aSaccharomyces cerevisiaecell, or a cell from another single-celled organism. It may also be a cell from a multi-cellular organism grown in culture. Some human cell lines that may be used for cell culture include DU145, H295R, HeLa, KBM-7, LNCaP, MCF-7, MDA-MB-468, PC3, SaOS-2, SH-SY5Y, T47D, THP-1, U87, and National Cancer Institute's 60 cancer cell line panel (NCI60).

The cell500may contain a dsDNA molecule502that has a first target site504. The cell500may also contain a first enzyme506that is configured to create a DSB at a cut site within the first target site504. For example, the first enzyme506may be a CRISPR/Cas system comprising a gRNA508that includes a spacer region (also called a proto-spacer element or targeting sequence) of about 20 nt that is complementary to one strand of the dsDNA502at the first target site504.

The dsDNA molecule502may also include a promoter510and a gene encoding a homology repair template512such as homology repair template514shown in this figure.

The dsDNA molecule502may be a vector or plasmid introduced to the cell500by any suitable method. A “vector” is a polynucleotide molecule, such as a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast or virus, into which a polynucleotide can be inserted or cloned. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g. retroviruses, replication defective retroviruses, lentiviruses, replicative defective lentiviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as “expression vectors.” Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. Plasmids suitable for expressing embodiments of the present invention, methods for inserting nucleic acid sequences into a plasmid, and methods for delivering recombinant plasmids to cells of interest are known in the art.

A vector may contain one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors), or be integrable with the genome of the defined host such that the cloned sequence is reproducible (e.g., non-episomal mammalian vectors). Accordingly, the vector can be an autonomously replicating vector, i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome. The vector can contain any means for assuring self-replication. Alternatively, the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Such a vector may comprise specific sequences that allow recombination into a particular, desired site of the host chromosome. A vector system can comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can include a reporter gene, such as a green fluorescent protein (GFP), which can be either fused in frame to one or more of the encoded polypeptides, or expressed separately. The vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants.

Several aspects of the invention relate to vector systems comprising one or more vectors, or vectors as such. Vectors can be designed for expression of transcripts (e.g. nucleic acid transcripts, proteins, or enzymes) in prokaryotic or eukaryotic cells. For example, transcripts can be expressed in bacterial cells such asEscherichia coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel,Gene Expression Technology: Methods In Enzymology,185, Academic Press. San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Vectors may be introduced and propagated in a prokaryote. In some embodiments, a prokaryote is used to amplify copies of a vector to be introduced into a eukaryotic cell or as an intermediate vector in the production of a vector to be introduced into a eukaryotic cell (e.g. amplifying a plasmid as part of a viral vector packaging system). Expression of proteins in prokaryotes is most often carried out inE. coliwith vectors containing constitutive or inducible promoters directing the expression of proteins. Examples of suitable inducibleE. coliexpression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al.,Gene Expression Technology: Methods In Enzymology185, Academic Press, San Diego, Calif. (1990) 60-89).

In some embodiments, a vector is capable of driving expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,Molecular Cloning: A Laboratory Manual.2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

Appropriate DNA segments may be inserted into a vector by a variety of procedures. In general, DNA sequences may be inserted into an appropriate restriction endonuclease site(s) by procedures known in the art, which may be performed without undue experimentation by a skilled artisan. A DNA segment in an expression vector may be operatively linked to an appropriate expression control sequence(s) (i.e., a promoter such as510) to direct synthesis. As used herein, a “promoter” is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3′ direction) coding or non-coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes. Various promoters, including inducible promoters, may be used to drive the various vectors of the present invention. A promoter may also contain sub-regions at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. Promoters may be constitutive, inducible, activatable, repressible, tissue-specific or any combination thereof.

Promoters may include any promoter known in the art for expression either in vivo or in vitro. Promoters which may be used in embodiments of the present invention may include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter may direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g. liver, pancreas), or particular cell types (e.g. lymphocytes). The promoters which may be used in embodiments of the present invention may also be inducible, such that expression may be decreased or enhanced or turned “on” or “off.” For example, promoters which respond to a particular signal (e.g., small molecule, metabolite, protein, molecular modification, ion concentration change, electric charge change, action potential, radiation, UV, and light) may also be used. Additionally, a tetracycline-regulatable system employing any promoter such as, but not limited to, the U6 promoter or the H1 promoter, may be used. By way of example and not of limitation, promoters which respond to a particular stimulus may include, e.g., heat shock protein promoters, and Tet-off and Tet-on promoters.

A promoter can be a constitutively active promoter (i.e., a promoter that is constitutively in an active/“ON” state), it may be an inducible promoter (i.e., a promoter whose state, active/“ON” or inactive/“OFF”, is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein.), it may be a spatially restricted promoter (i.e., transcriptional control element, enhancer, etc.)(e.g., tissue specific promoter, cell type specific promoter, etc.), and it may be a temporally restricted promoter (i.e., the promoter is in the “ON” state or “OFF” state during specific stages of embryonic development or during specific stages of a biological process, e.g., hair follicle cycle in mice).

A promoter drives expression or drives transcription of the nucleic acid sequence that it regulates. Herein, a promoter is considered to be “operably linked” when it is in a correct functional location and orientation in relation to a nucleic acid sequence it regulates to control (“drive”) transcriptional initiation and/or expression of that sequence.

A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5′ non-coding sequences located upstream of the coding segment of a given gene or sequence. Such a promoter is referred to as an “endogenous promoter.”

In some embodiments, a coding nucleic acid sequence may be positioned under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the encoded sequence in its natural environment. Such promoters may include promoters of other genes; promoters isolated from any other cell; and synthetic promoters or enhancers that are not “naturally occurring” such as, for example, those that contain different elements of different transcriptional regulatory regions and/or mutations that alter expression through methods of genetic engineering that are known in the art. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including polymerase chain reaction (PCR). Contemplated herein, in some embodiments, are RNA pol II and RNA pol III promoters. Promoters that direct accurate initiation of transcription by an RNA polymerase II are referred to as RNA pol II promoters. Examples of RNA pol II promoters for use in accordance with the present disclosure include, without limitation, human cytomegalovirus promoters, human ubiquitin promoters, human histone H2A1 promoters and human inflammatory chemokine CXCL 1 promoters. Other RNA pol II promoters are also contemplated herein. Promoters that direct accurate initiation of transcription by an RNA polymerase III are referred to as RNA pol III promoters. Examples of RNA pol III promoters for use in accordance with the present disclosure include, without limitation, a U6 promoter, a HI promoter and promoters of transfer RNAs, 5S ribosomal RNA (rRNA), and the signal recognition particle 7SL RNA.

Illustrative promoters include, but are not limited to the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), an enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep. 1; 31(17)), a human H1 promoter (H1), and the like.

Examples of inducible promoters include, but are not limited to T7 RNA polymerase promoter, T3 RNA polymerase promoter, Isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, lactose induced promoter, heat shock promoter, Tetracycline-regulated promoter, Steroid-regulated promoter, Metal-regulated promoter, estrogen receptor-regulated promoter, etc. Inducible promoters can therefore be regulated by molecules including, but not limited to, doxycycline; RNA polymerase, e.g., T7 RNA polymerase; an estrogen receptor; an estrogen receptor fusion; etc. Cells, such as cells in culture, may be transfected or transformed with the dsDNA molecule502. Transfection is the process of deliberately introducing naked or purified polynucleotides into eukaryotic animal cells. Transformation refers to DNA transfer in bacteria and non-animal eukaryotic cells, including plant cells. Transfection may be performed using viruses or mechanical methods. Viral transfection introduces foreign DNA into a cell by a virus or viral vector. Transfection with a virus may introduce the DNA into the genome of the host cell. Mechanical transfection typically involves opening transient pores or “holes” in the cell membrane to allow the uptake of material. Transfection can be carried out using calcium phosphate (i.e. tricalcium phosphate), by electroporation, microinjection, gene gun, impalefection, hydrostatic pressure, continuous infusion, sonication, lipofection, nanoparticles containing the dsDNA molecule502(e.g., mesoporous silica nanoparticles or gold nanoparticles) or by mixing a cationic lipid with the material to produce liposomes which fuse with the cell membrane and deposit their cargo inside. Nanoparticles used to introduce foreign DNA may be ionically charged or have targeting ligands to deliver to specific cells or sites.

One viral transfection technique for transferring genetic material to hard-to-transfect cells is recombinant adeno-associated virus (AAV) delivery. This is a type of viral transduction that does not integrate into the host genome. AAV-based systems have been used successfully to introduce the gene forS. pyogenesCas9 (SpCas9) together with its optimal promoter and polyadenylation signal using the AAVpro CRISPR/Cas9 Helper Free System (AAV2) available from Takara Bio USA, Inc.

Conjugation may also be used to introduce the dsDNA molecule502into a cell. Although conjugation in nature occurs more frequently in bacteria, transfer of genetic material from bacterial to mammalian cells is also possible. See Waters V. L.,Conjugation between bacterial and mammalian cells.29 (4) Nature Genetics 375 (2001).

The cell500may also include a gene516under the control of a promoter518and an operator520. The gene516may encode a ssRNA sequence522comprising a 3′-end sequence524and a 5′-end sequence526. AN HDR template514may be generated from the gene516. In one implementation, the HDR template514is the ssRNA sequence522itself. The 3′-end sequence524and the 5′-end sequence526are complementary to one strand of a dsDNA molecule502over at least part of a target site504. Homology between the 3′-end sequence524and the 5′-end sequence526allows the ssRNA sequence522to hybridize with portions of the dsDNA on either side of a DSB created at a cut site in the target site504.

In implementations in which the gene516directly encodes the HDR template514, the gene516will encode a cut site528that may be cut by an enzyme such as the first enzyme506. Unless protected from the enzyme, the cut site528in the gene516may be unintentionally cut when the enzyme contacts the gene516.

One technique for protecting the cut site528from the first enzyme506is physical separation. In a cell-free system, such as one that uses microfluidics, the gene516may be maintained in one chamber and the ssRNA sequence522may be moved from the chamber containing the gene516into a different chamber where the enzyme506is present.

Physical separation may also be used in cellular implementations. The gene516and the enzyme506may be contained in different cellular chambers. In one implementation, the gene516may be in the nucleus and the enzyme may be outside the nucleus in the cytoplasm or in another cellular chamber. The gene516may remain in the nucleus if it is part of the cell's genome. A nuclear export signal (NES) may be used to keep the enzyme, or other component of the system, out of the nucleus. A NES is a short amino acid sequence of four hydrophobic residues in a protein that targets it for export from the cell nucleus to the cytoplasm through the nuclear pore complex using nuclear transport. Similarly, a nuclear localization signal (NLS) may be used to keep the enzyme in the nucleus. A NLS is an amino acid sequence that tags a protein for import into the cell nucleus by nuclear transport. Typically, a NLS consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface. Different nuclear localized proteins may share the same NLS. An NLS has the opposite function of a NES. Persons of ordinary skill in the art will be able to modify or engineer a protein such as a nuclease or other enzyme to include a NES or a NLS.

The physical location of RNA in a cell may also be controlled. The ssRNA sequence522may be exported from its site of transcription in the nucleus to the cytoplasm or other destination outside the nucleus where the enzyme is present. RNA export is described in Sean Carmody and Susan Wente,mRNA Nuclear Export at a Glance,122 J. of Cell Science 1933 (2009) and Alwin Kohler and Ed Hurt,Exporting RNA from the Nucleus to the Cytoplasm,8 Nature Reviews Molecular Cell Biology 761 (2007).

Splicing may be used in place of or in addition to physical separation to protect the gene516from being cut by the enzyme506. In one implementation, the gene516may include a sequence with a portion that is later removed by splicing. This additional portion changes the sequence of nucleotides in the gene516so that there is no cut site528present. The ssRNA sequence522will becomes an HDR template514through splicing, which also introduces the cut site528.

Alternative splicing, or differential splicing, is a regulated process during gene expression that results in a single gene coding for multiple proteins. In this process, particular exons of a gene may be included within or excluded from the final, processed messenger RNA (mRNA) produced from that gene. Consequently, the proteins translated from alternatively spliced mRNAs will contain differences in their amino acid sequence and, often, in their biological functions. The production of alternatively spliced mRNAs is regulated by a system of trans-acting proteins that bind to cis-acting sites on the primary transcript itself. Such proteins include splicing activators that promote the usage of a particular splice site, and splicing repressors that reduce the usage of a particular site. There are multiple types of alternative splicing including exon skipping, mutually exclusive exons, alternative donor sites, alternative acceptor sites, and intron retention. Exon skipping is one way to cause splicing in the ssRNA sequence522; in this case, an exon may be spliced out of the primary transcript. Persons having ordinary skill in the art will understand how to design the gene516so that it includes a splice site at a specified location. Alternative splicing may be implemented as a technique to prevent creation of a DSB in the gene516even if the gene516and enzyme506are not physically separated.

Self-excising elements may function similarly to splicing. The gene516may be designed to include a region that, when transcribed into RNA, includes one or more self-excising elements. Inclusion of the self-excising elements, for example in a way that disrupts the cut site528, prevents the gene516from being recognized by the enzyme and the excision converts the ssRNA sequence522into the HDR template514. One type of self-excising elements are ribozymes, which are RNA enzymes that function as reaction catalysts. Ribozymes are RNA sequences that catalyze a (trans-esterification) reaction to remove the ribozyme sequence itself from the rest of the RNA sequence. Essentially these are considered introns, which are intragenic regions spliced from mRNA to produce mature RNA with a continuous exon (coding region) sequence. Self-excising introns/ribozymes consist of group I and group II introns. Many group I introns in bacteria are known to self-splice and maintain a conserved secondary structure comprised of a paired element which uses a guanosine (GMP, GDP, or GTP) cofactor. An example of a group I intron is theStaphylococcusphage twort.ORF143. Group I and group II introns are considered self-splicing because they do not require proteins to initialize the reaction. Self-excising sequences are known and one of ordinary skill in the art will understand how to include a self-excising sequence in the gene516. Aspects of self-excising ribozymes are shown inIn Vivo Protein Fusion Assembly Using Self Excising Ribozymeavailable at http://2011.igem.org/Team:Waterloo (last visited Mar. 3, 2017).

A series of homologous bridges may also be used to generate a recombinant sequence that is the gene template for the ssRNA sequence522. The homologous bridges may be present in the DNA at various, separate locations so that the gene516does not include a cut site528. This technique is also known as multi-fragment cloning or extension cloning. The final HDR template514is made up of transcripts of the multiple overlapping segments. One suitable technique for combining the multiple-overlapping fragments into the HDR template514is Sequence and Ligation-Independent Cloning (SLIC). This technique is described in Mamie Li and Stephen Elledge,Harnessing Homologous Recombination in vitro to Generate Recombinant DNA Via SLIC,4 Nature Methods 250 (2007). Another suitable technique for joining multiple-overlapping fragments is provided by Jiayuan Quan and Jingdong Tian,Circular Polymerase Extension of Cloning of Complex Gene Libraries and Pathways,4(7) PLoS ONE e6441 (2009).

Methylation may be used to protect HDR templates from premature cutting by restriction enzymes because some restriction enzymes do not cut methylated DNA. Other nucleases such as Cas9 may also be prevented from cutting by methylation of a cutting region or PAM recognition site. DNA methylation is a process by which methyl groups are added to the DNA molecule. Methylation can change the activity of a DNA segment without changing the sequence. Two of DNA's four bases, cytosine and adenine, can be methylated. A methylase is an enzyme that recognizes a specific sequence and methylates one of the bases in or near that sequence. Methylation may be controlled by epigenetic editing using a targeting device that is a sequence-specific DNA binding domain which can be redesigned to recognize desired sequences. The targeting device may be fused to an effector domain, which can modify the epigenetic state of the targeted locus. Techniques for using epigenetic editing will be understood by one of ordinary skill in the art. Epigenome manipulations are described in Park, et al.,The epigenome: the next substrate for engineering.17 Genome Biology 183 (2016). HDR templates made of RNA may also be modified by methylation. S. Lin and R. Gregory,Methyltransferases modulate RNA stability in embryonic stem cells,16(2) Nature Cell Biology 129 (2014).

In one implementation, the HDR template514is a ssDNA sequence complementary to the ssRNA sequence522. The ssDNA sequence may be created by reverse transcriptase reading (RT) the ssRNA sequence522and synthesizing a complementary ssDNA sequence. RT is an enzyme used to generate cDNA from an RNA template, a process termed reverse transcription. RT is widely used in the laboratory to convert RNA to DNA for use in procedures such as molecular cloning, RNA sequencing, PCR, and genome analysis. RT enzymes are widely available from multiple commercial sources. Procedures for use of RT is well known to those of ordinary skill in the art.

The 3′-end sequence530and the 5′-end sequence532of the HDR template514are homologous to one strand of the dsDNA502over at least a portion of the first target site504. The HDR template514, in both ssDNA and ssRNA implementations, includes a middle portion534that, when incorporated into the dsDNA502, acts as a record on a signal detected by the engineered signaling pathway536. In an implementation, the middle portion534also introduces another target site as described elsewhere in this disclosure.

Enzyme506is illustrated here as a CRISPR/Cas complex with gRNA508. Other types of enzymes discussed above may be used instead of the CRISPR/Cas complex. The single-stranded tail of the gRNA508may be extended with a sequence complementary to all or part of the HDR template514. The HDR template514may partially hybridize to the tail of the gRNA508forming a double-stranded region538. This brings a copy of the HDR template514into close physical proximity with the location of the DSB created by the CRISPR/Cas complex506which can increase HDR efficiency.

The extended tail of the gRNA508may also be designed so that it matches the binding domain of a transcription activator-like effector (TALE) protein. The TALE protein may also have a binding domain complementary to the HDR template514. This will also bring the HDR template into close proximity with the location of the DSB. The tail of the gRNA508may be extended to create regions for attachment of multiple copies of the HDR template514or TALE proteins.

TALE proteins are proteins secreted byXanthomonasbacteria via their type III secretion system when the bacteria infect various plant species. These proteins can bind promoter sequences in the host plant and activate the expression of plant genes that aid bacterial infection. They recognize plant DNA sequences through a central repeat domain consisting of a variable number of about 34 amino acid repeats. There appears to be a one-to-one correspondence between the identity of two critical amino acids in each repeat and each DNA base in the target site. The most distinctive characteristic of TAL effectors is a central repeat domain containing between 1.5 and 33.5 repeats that are usually 34 nt in length (the C-terminal repeat is generally shorter and referred to as a “half repeat”). A typical repeat sequence may be shared across many TALE proteins but the residues at the 12thand 13thpositions are hypervariable (these two amino acids are also known as the repeat variable diresidue or RVD). This simple correspondence between amino acids in TAL effectors and DNA bases in their target sites makes them useful for protein engineering applications.

Subsequent to creation of a DSB in the target site504, the molecule538that has hybridized to the tail of the gRNA508may be released. In some implementations, introduction of a nucleotide sequence complementary to the tail of the gRNA508or binding domain of the TALE protein may compete with the attached molecule538and cause disassociation of the HDR template514, TALE protein, or other molecule. This competition may cause the HDR template514to become available for binding to the dsDNA502on either side of the DSB.

The cell500may also include one or more engineered signaling pathways536. As used herein, “engineered signaling pathway” includes any pathway in which at least one portion of the pathway is intentionally modified with molecular biology techniques to be different from the wild type pathway and a signal (intracellular or extracellular) causes a change in a rate of transcription of a gene. The engineered signaling pathway536may induce a promoter such as the promoter512described above. The engineered signaling pathway536may also cause a transcription factor to bind to an operator such as the operator514described above and prevent transcription. In one implementation, the gene affected by the engineered signaling pathway536may be the gene516that encodes for the ssRNA sequence522. Thus, the engineered signaling pathway536may function to control an amount of the HDR template514available in the cell500. In one implementation, the gene affected by the engineered signaling pathway536may encode for an enzyme that creates DSBs in dsDNA such as enzyme506. Thus, the number of enzymes which create DSBs in the target sites504may be regulated by the engineered signaling pathway536. The engineered signaling pathway536may control the transcription of genes that encode other proteins associated with HDR.

The cell500may include multiple different engineered signaling pathways536each responding to a unique signal and each promoting or repressing expression of genes responsible for the creation of the HDR templates522and/or enzymes506. Thus, intracellular or extracellular signals may be used to vary the levels of HDR templates514and/or enzymes506in the cell500thereby changing which target sites504are cut and which sequences are used to repair DSBs through HDR. Responding by up or down regulating any of multiple promoters and/or operators allows the cell500to record a log in its DNA of events and complex interactions of events sensed by engineered signaling pathways. In one implementation, the engineered signaling pathway536may include an external receptor540that can detect extracellular signals across a membrane542. The membrane542may be a cell wall, lipid bilayer, artificial cell wall, or synthetic membrane.

The cell500may also include one or more additional dsDNA molecules544that may include a second target site546. Similar to the first dsDNA molecule502, the additional dsDNA molecule544may include only a single instance of the second target site546. Alternatively, the additional dsDNA molecule544may include multiple copies of the same target site or multiple different target sites. The additional dsDNA molecule544may be introduced to the cell500by any of the techniques described above. In some implementations, the first dsDNA molecule502and the additional dsDNA molecule544may be introduced by the same procedure. A ratio of the first dsDNA molecule502and the additional dsDNA molecule544in the cell500may be controlled by regulating the respective copies of the dsDNA molecules added to the cell500.

The additional dsDNA molecule544and the second target site546may have identical or similar sequences to the first dsDNA molecule502and the first target site504. Thus, the additional dsDNA molecule544may be thought of as a “copy” of the first dsDNA molecule502in some implementations. This additional copy of an identical or similar molecule may provide redundancy by creating a second log that, absent errors, will record the same series of events in both dsDNA molecules502,544. In one implementation, the additional dsDNA molecule544may include a target site546with a different sequence than the first target site504in the first dsDNA molecule502. Having different target sites504,546in different dsDNA molecules502,544allows for simultaneous, or alternating, encoding of binary data in two different encoding schemes. The two different encoding schemes may be non-overlapping or “orthogonal” so that the enzymes and HDR templates associated with one encoding scheme do not interact with the dsDNA molecule used for the other encoding scheme. For example, insertion of DNA into the first target site504may record the presence of signals related to temperature and insertion of DNA into the second target site546may record the presence of signals related to light levels. It is understood, that in actual implementation there may be many hundreds or thousands of dsDNA molecules with respective target sites. There may also be a corresponding number of different encoding schemes and different sequences for the respective target sites for creating a detailed log of multiple different signals.

In an implementation, the additional dsDNA molecule544may include an operon548that encodes components used for logging molecular events. An operon is a contiguous region of DNA that includes cis-regulatory regions (e.g., repressors, promoters) and the coding regions for one or more genes or functional mRNAs (e.g., siRNA, tracrRNA, gRNA, shRNA, etc). The operon548may be delivered in a circular vector, such as the additional dsDNA molecule544, or may be inserted into genomic DNA of the cell500through gene editing techniques known to those of skill in the art. In an implementation, the operon548may include genes encoding all of the components used by the cell500for performing HDR. Thus, addition of a vector such as the dsDNA molecule544may enable a cell500that includes the necessary engineered signaling pathway536to respond to detected signals by adding homology repair templates522into a target site546on the added dsDNA molecule544. In this implementation, the homology repair template514, the enzyme506, and any accessory proteins may be supplied by genes included in the operon548. The genes in the operon548may be under the control of a single promoter550and operator552.

In an implementation, the operon548may include any or all of a gene encoding an HDR template554, a gene encoding an enzyme configured to make DSBs556, and a gene that encodes a tracking molecule558(e.g., RNA, DNA, or protein) for monitoring “state” as described below. An operon548that includes genes encoding all of the products for performing HDR may be added to a cell-free system on a circular dsDNA molecule544that also includes a target site546to provide complete instructions for a molecular event logging system on one molecule.

The term “operably linked” as used herein means placing a gene under the regulatory control of a promoter, which then controls the transcription and optionally the translation of the gene. In the construction of heterologous promoter/structural gene combinations, it is generally preferred to position the genetic sequence or promoter at a distance from the gene transcription start site that is approximately the same as the distance between that genetic sequence or promoter and the gene it controls in its natural setting; i.e. the gene from which the genetic sequence or promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting; i.e., the genes from which it is derived. “Constitutive promoters” are typically active, i.e., promote transcription, under most conditions. “Inducible promoters” are typically active only under certain conditions, such as in the presence of a given molecule factor (e.g., IPTG) or a given environmental condition (e.g., particular CO2concentration, nutrient levels, light, heat). In the absence of that condition, inducible promoters typically do not allow significant or measurable levels of transcriptional activity. For example, inducible promoters may be induced according to temperature, pH, a hormone, a metabolite (e.g., lactose, mannitol, an amino acid), light (e.g., wavelength specific), osmotic potential (e.g., salt induced), a heavy metal, or an antibiotic. Numerous standard inducible promoters are known to one of skill in the art.

Illustrative eukaryotic promoters known to one of skill in the art are listed below.

PrimarilyPromoterused forDescriptionAdditional considerationsCMVGeneralStrong mammalianMay contain an enhancer region. Can beexpressionexpression promotersilenced in some cell types.from the humancytomegalovirusEF1aGeneralStrong mammalianTends to give consistent expression regardlessexpressionexpression fromof cell type or physiology.human elongationfactor 1 alphaSV40GeneralMammalian expressionMay include an enhancer.expressionpromoter from thesimian vacuolatingvirus 40PGK1GeneralMammalian promoterWidespread expression, but may vary by cell(human orexpressionfrom phosphoglyceratetype. Tends to resist promoter down regulationmouse)kinase gene.due to methylation or deacetylation.UbcGeneralMammalian promoterAs the name implies, this promoter isexpressionfrom the humanubiquitous.ubiquitin C genehumanGeneralMammalian promoterUbiquitous. Chicken version is commonlybeta actinexpressionfrom beta actin geneused in promoter hybrids.CAGGeneralStrong hybridContains CMV enhancer, chicken beta actinexpressionmammalian promoterpromoter, and rabbit beta-globin spliceacceptor.TREGeneralTetracycline responseTypically contains a minimal promoter withexpressionelement promoterlow basal activity and several tetracyclineoperators. Transcription can be turned on oroff depending on what tet transactivator isused.UASGeneralDrosophilapromoterRequires the presence of Gal4 gene to activateexpressioncontaining Gal4promoter.binding sitesAc5GeneralStrong insect promoterCommonly used in expression systems forexpressionfromDrosophilaActinDrosophila.5c genePolyhedrinGeneralStrong insect promoterCommonly used in expression systems forexpressionfrom baculovirusinsect cells.CaMKIIaGeneCa2+/calmodulin-Used for neuronal/CNS expression. Modulatedexpressiondependent proteinby calcium and calmodulin.forkinase II promoteroptogeneticsGAL1, 10GeneralYeast adjacent,Can be used independently or together.expressiondivergently transcribedRegulated by GAL4 and GAL 80.promotersTEF1GeneralYeast transcriptionAnalogous to mammalian EF1a promoter.expressionelongation factorpromoterGDSGeneralStrong yeastVery strong, also called TDH3 or GAPDH.expressionexpression promoterfrom glyceraldehyde 3-phosphagedehydrogenaseADH1GeneralYeast promoter forFull length version is strong with highexpressionalcohol dehydrogenase Iexpression. Truncated promoters areconstitutive with lower expression.CaMV35SGeneralStrong plant promoterActive in dicots, less active in monocots, withexpressionfrom the Cauliflowersome activity in animal cells.Mosaic VirusUbiGeneralPlant promoter fromGives high expression in plants.expressionmaize ubiquitin geneH1smallFrom the humanMay have slightly lower expression than U6.RNApolymerase III RNAMay have better expression in neuronal cells.expressionpromoterU6smallFrom the human U6Murine U6 is also used, but may be lessRNAsmall nuclear promoterefficient.expression

Illustrative prokaryotic promoters known to one of skill in the art are listed below.

FIG. 6shows a dsDNA molecule600that contains genes for encoding the components used to incorporate an HDR template into a target site. The dsDNA molecule600may be the same or similar to the dsDNA molecule502or544introduced inFIG. 5. In an implementation, the dsDNA molecule600may be a vector or plasmid as described above. This dsDNA molecule600may be added to a cell by any of the techniques discussed in this disclosure such as transfection, transformation, conjugation, etc.

In an implementation, the dsDNA molecule600may encode one or more target sites for insertion of HDR templates such as a first target site602and a second target site604. HDR templates integrated into the target sites602,604may correspond to timing signals or molecular events experienced by the cell. The dsDNA molecule600can also encode multiple operons each including a promoter, an operator, and a gene. A gene encoding an HDR template606may be regulated by a promoter608and an operator610. A gene encoding a Cas9 enzyme612may be regulated by a second promoter614and second operator616. Cas9 is shown in this example but any enzyme capable of creating DSBs may be alternatively present in the dsDNA molecule600. Cas9 targets a specific sequence for cutting based on the associated gRNA and the dsDNA molecule600may also include a gene for a gRNA618regulated by a third promoter620and a third operator622. In this example, the HDR template606, the nuclease Cas9612, and the gRNA618are all controlled by different sets of promoters and operators. However, any two or all three may be combined together in a single operon controlled by the same regulatory sequences.

Insertion of an HDR template in response to a timing signal occurs when there is a DSB at an appropriate target site and sufficient copies of the HDR template are available to contact and recombine with the cut polynucleotide at the target site. Thus, the ability to integrate an HDR template in response to a timing signal may be regulated by controlling availability of the HDR template or the enzyme used to create the DSB at the appropriate target site. Addition of the dsDNA molecule600to a cell provides all of the components for recording timing signals. Presence of a timing signal whether due to manual manipulation of the cell or due to a periodic cycle within the cell can affect any of the promoters608,614,620or operators610,616,622. Thus, integration of an HDR template into a target site602,604can be regulated by controlling expression of any of the gene for the HDR template606, the gene for the Cas9 nuclease612, or the gene for the gRNA618. The regulation may include inducing expression of one of the promoters608,614,620, ceasing inhibition of one of the operators610,616,622, or otherwise manipulating the regulatory elements associated with one of the relevant genes.

FIG. 7shows a diagram700including a molecule702that degrades in a cellular system at a known rate. The change in concentration of the molecule702due to degradation provides a source of timing that can be used to determine the timing of molecular events detected by the cellular system. The molecule702may be any one of a number of different types of molecules that are involved in repair of a DSB by using an HDR template.

For example, the molecule702may be all or part of an enzyme704configured to create a DSB in a double-stranded polynucleotide such as the double-stranded polynucleotide704. The enzyme706may be any of the nucleases discussed above such as Cas9. Proteins such as Cas9 will degrade in cells due to the presence of proteases. This rate of degradation may be experimentally established for a given cellular system. For in vivo logging, it is possible to use externally pre-synthesized, purified Cas9 or include the gene for Cas9 in a vector or plasmid as shown inFIG. 6. As discussed above, the enzyme706creates a DSB at a cut site that is flanked by regions homologous to an HDR template. Hybridization of the homologous regions to the HDR template permits homologous directed repair of the DSB. The enzyme706is a protein; the stability of a protein can be altered by introducing mutations into the amino acid sequence that make the protein more or less resistant to denaturation or proteolytic degradation. Persons having ordinary skill in the art will appreciate various techniques to modify the duration that a protein remains active in a given cellular environment. Techniques such as directed evolution, DNA shuffling and two-hybrid screening are known in the art and may be used to rapidly screen large numbers of mutant proteins for the desired stability characteristics. In addition, protein degradation rate may be altered by attaching a short, organism-specific, oligonucleotide sequence to the 3′-end of the gene which encodes the protein as described in Andersen et al. (1998) Appl. Environ. Microbiol. 64:2240-2246. This sequence targets the encoded protein for rapid degradation by the cell. Thus, the rate of degradation of a protein may be adjusted to achieve a desired timing.

In an implementation, the molecule702may be an HDR template708. As described above, HDR template708may be ssDNA or ssRNA. Natural degradation processes in a cell can cause free ssDNA or mRNA to degrade over time. The degradation speed may be increased by addition of proteasomes or nucleases. Thus, intentional design of the cellular system including the amount of proteases and/or nucleases may be used to tune or adjust the rate of degradation.

In an implementation, the molecule702can be a transcription factor710. The transcription factor710can increase transcription of a gene encoding the HDR template708or a gene encoding enzyme configured to create a DSB in the double-stranded polynucleotide704. For example, the transcription factor710may interact with any of the promoters608,614,620described inFIG. 6.

The molecule702can be a RNA molecule712. For example, the RNA molecule712may be gRNA that functions with the Cas9 nuclease. Cas9 is able to take up the gRNA dynamically when available, thus the Cas9 enzyme may be present in abundance and the ability to create targeted DSBs may depend on the concentration of gRNA. After export to the cytoplasm, mRNA (including gRNA) is protected from degradation by a 5′ cap structure and a 3′ poly(A) tail. The rate of mRNA degradation is typically minutes in prokaryotes and hours-months in eukaryotes. A rate of degradation of the RNA712may be determined in part by a modification to a 3′ poly(A) tail. A longer poly(A) tail generally correlates with greater stability of RNA and a shorter poly(A) tail generally leads to faster degradation of the RNA. Specifically, the degradation of RNA may be affected by the 3′-untranslated region (3′-UTR). The 3′-UTR can influence polyadenylation, translation efficiency, localization, and stability of the mRNA. The 3′-UTR contains both binding sites for regulatory proteins as well as microRNAs (miRNAs). By binding to specific sites within the 3′-UTR, miRNAs can decrease gene expression of various mRNAs by either inhibiting translation or directly causing degradation of the transcript. The 3′-UTR contains both binding sites for regulatory proteins as well as miRNAs. By binding to specific sites within the 3′-UTR, miRNAs can decrease gene expression of various mRNAs by either inhibiting translation or directly causing degradation of the transcript.

Mature microRNAs (miRNAs) are a class of naturally occurring, small non-coding RNA molecules, about 21-25 nt in length. They are found in plants, animals and some viruses, and have functions in RNA silencing and post-transcriptional regulation of gene expression. MicroRNAs are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to downregulate gene expression in a variety of manners, including translational repression, mRNA cleavage, and deadenylation.

Encoded by eukaryotic nuclear DNA in plants and animals and by viral DNA in certain viruses whose genome is based on DNA, miRNAs function via base-pairing with complementary sequences within mRNA molecules. As a result, the RNA712can be silenced, by one or more of the following processes: cleavage of the RNA712strand into two pieces, destabilization of the RNA712through shortening of its poly(A) tail, and less efficient translation of the RNA712into proteins by ribosomes. For example, miR16 contains a sequence complementary to the AU-rich element found in the 3′-UTR of many unstable mRNAs, such as TNF alpha or GM-CSF. It has been demonstrated that given complete complementarity between the miRNA and target mRNA sequence, Ago2 can cleave the mRNA and lead to direct mRNA degradation. Jing, Q. et al.Involvement of microRNA in AU-rich element-mediated mRNA instability.120(5) Cell 623 (2005).

The genes encoding miRNAs are much longer than the processed mature miRNA molecule. Many miRNAs are known to reside in introns of their pre-mRNA host genes and share their regulatory elements, primary transcript, and have a similar expression profile. MicroRNAs are transcribed by RNA polymerase II as large RNA precursors called pri-miRNAs and comprise of a 5′ cap and poly-A tail3. The pri-miRNAs are processed in the nucleus by the microprocessor complex, consisting of the RNase III enzyme Drosha4, and the double-stranded-RNA-binding protein, Pasha/DGCR85. The resulting pre-miRNAs are approximately 70-nt in length and are folded into imperfect stem-loop structures. The pre-miRNAs are then exported into the cytoplasm by the karyopherin exportin 5 (Exp5) and Ran-GTP complex. Ran (ras-related nuclear protein) is a small GTP binding protein belonging to the RAS superfamily that is essential for the translocation of RNA and proteins through the nuclear pore complex. The Ran GTPase binds Exp5 and forms a nuclear heterotrimer with pre-miRNAs. Once in the cytoplasm, the pre-miRNAs undergo an additional processing step by the RNAse III enzyme Dicer9 generating the miRNA, a double-stranded RNA approximately 22 nt in length.

Concentration of the molecule702may vary in the cellular system with respect to time. Starting from an initial concentration, degradation of the molecule702will eventually cause the concentration of the molecule702to drop below a threshold level714. The threshold level714may be determined experimentally or known from the behavior of the molecule702. If the molecule702functions to promote insertion of the HDR template708into the double-stranded polynucleotide704, then insertion of the HDR template708into a target site716of the double-stranded polynucleotide704indicates that the incorporation occurred prior to the concentration of the molecule702dropping below the threshold level714. Alternatively, if the molecule702functions as a repressor, for example a transcription factor710that binds to an operator, then incorporation of the HDR template708will indicate that enough time has passed such that the concentration of the molecule702has dropped below the threshold level714.

This technique for controlling insertion of the HDR template708into the target site716of a double-stranded polynucleotide704based on the concentration of a molecule702with respect to a threshold level714may be combined with any of the other techniques described in this disclosure to control the timing of HDR based on the rate of degradation of the molecule702.

Illustrative Processes

For ease of understanding, the processes discussed in this disclosure are delineated as separate operations represented as independent blocks. However, these separately delineated operations should not be construed as necessarily order dependent in their performance. The order in which the process is described is not intended to be construed as a limitation, and any number of the described process blocks may be combined in any order to implement the process, or an alternate process. Moreover, it is also possible that one or more of the provided operations may be modified or omitted.

Process800shows an illustrative technique for introducing timing indicators into the DNA of a cell based on manual manipulation of the cell.

At802, the cell is exposed to a timing indicator at a known time. The timing indicator may be a chemical or environmental condition that causes the cell to integrate an HDR template into a double-stranded polynucleotide. Exposure to the timing indicator may be performed “manually” by placing the cell in an environment which is known to generate the corresponding signal. For example, the cell could be bathed in a solution containing a particular chemical which causes a response in a signaling pathway of the cell. Because exposure is controlled, the time of exposure may be recorded so that later analysis can identify insertion of this first HDR template as having occurred at the time of exposure.

The exposure may include making sufficient copies of the first HDR template available so that HDR can occur, making a nuclease available, if the nuclease is Cas9, then the exposure may include making targeted gRNA available. The timing indicator, no matter how generated, may be detected by an engineered signaling pathway in the cell and this detection may cause the cell to increase transcription of either the first HDR template or an enzyme. Increasing transcription of the first HDR template ultimately results in more copies of the first HDR template being available for incorporation into the double-stranded polynucleotide in the cell. The first HDR template includes a first middle portion that is not homologous to the double-stranded polynucleotide, and thus, represents a new nucleotide sequence that will be inserted by homology directed repair. Similarly, increase in the number of functional enzymes that act at the cut site, increases the number of DSBs that are available to be repaired by the HDR templates. Either, or both, may ultimately result in more copies of the first middle portion of the first HDR template being incorporated into the double-stranded polynucleotide.

Engineered signaling pathways in the cell may be used to cause integration of HDR templates in response to light, temperature, change in pH, etc. Conditions of the cell may be controlled so that the trigger for integrating a time indicator does not occur unless intentionally caused. For example, the cells may be kept in the dark, the temperature may be tightly regulated, the pH may be maintained by buffering solution, etc.

The HDR temple may be generated by a gene under control of a regulated promoter that responds to the exposure. The mRNA gene product may be converted to DNA through use of RT to create a DNA molecule that is the final HDR template. In some implementations, the mRNA may itself serve as the HDR template without conversion to DNA.

In order to limit where the first enzyme cuts the double-stranded polynucleotide, the first target site may be unique in the double-stranded polynucleotide at the time of making the first DSB. The first target site may also be unique across a population of double-stranded polynucleotides that is available for the first enzyme to act on. For example, if there are multiple circular dsDNA molecules within a cell, the first target site may exist only once within the entire population of circular dsDNA molecules. Alternatively, the first target site may be unique per dsDNA molecule, but the first enzyme may have access to multiple different dsDNA molecules each including one instance of the first target site. It is understood by persons having ordinary skill in the art that the enzyme (even if referred to in the singular herein) may include a plurality of individual and equivalent enzyme molecules. In some implementations, the first target site may include a first subsequence that is repeated once resulting in a second subsequence that is the same as the first subsequence. For example, if the first subsequence is GTACTA then the second subsequence is the same and the sequence of the target site is GTACTAGTACTA (SEQ ID NO: 9).

The enzyme may be any of the illustrative types of enzymes identified in this disclosure such as a restriction enzyme, HE, a CRISPR/Cas system, a TALEN, or a zinc finger.

The HDR template may include a 3′-end sequence and a 5′-end sequence each encoding a second subsequence that is homologous to the first subsequence in the first target site. Thus, in this implementation the 3′-end sequence and the 5′-end sequence have the same sequence, but in other implementations they may have different sequences. The first HDR template may also include a middle portion that includes two adjacent instances of a third subsequence that forms the next target site after insertion into the double-stranded polynucleotide as shown inFIGS. 1 and 2.

This contact may result from diffusion and/or Brownian motion of the HDR template moving within the cell until it contacts the DSB on the double-stranded polynucleotide. Contacting the double-stranded polynucleotide with the first HDR template may involve movement of multiple copies of the first HDR template into a chamber that contains the double-stranded polynucleotide such as, for example, by a microfluidics system or localization to a cellular chamber such as the nucleus. In one implementation, contacting the double-stranded polynucleotide involves upregulating expression of a gene encoding for a RNA sequence that is itself the HDR template or that serves as a template for creation of the HDR template. Upregulating expression of the gene may include any of activating a promoter controlling transcription of the gene, upregulating the promoter controlling transcription, unblocking a promoter controlling transcription of the gene, and/or inhibiting action of a repressor or silencer.

At804, the cell logs one or more molecular events. The cell can integrate a second HDR template into a repair formed by the first HDR template in the double-stranded polynucleotide in response to a molecular event. The repair formed by the first HDR template includes the next target site into which the second HDR template may be integrated. Thus, in this example, the cell logs a molecular event in a portion of polynucleotide that is created by adding the first HDR template in response to the timing indicator. The molecular event may be an intracellular or extracellular event that corresponds to a change in a biological condition of the cell or a change in the condition of the external environment. The molecular event is different than the timing indicator at least because the timing of the molecular event is not manually controlled and the molecular event is initiated by a sensed condition. The cell can iteratively integrate multiple copies of the second HDR template into the double-stranded polynucleotide while the molecular event continues.

Using nomenclature introduced earlier in this disclosure, the first HDR template may be represented as XaXXaX which can be inserted into the target site XX and includes, after insertion, the same target site XX in the middle. While, the second HDR template may be represented as XbXXbX with “b” representing the part of the second middle portion that is different from the first middle portion of the first HDR template (i.e., “a”≠“b”). Thus, presence of the polynucleotide sequence corresponding to “a” corresponds to the timing indicator and presence of “b” corresponds to a molecular event. The sequence XaXbXXbXaX can then provide a record that molecular event “b” occurred at or shortly after the time point indicated by “a.” The sequences “a” and “b” both as they exist in HDR templates and following integration into a double-stranded polynucleotide are “identifier regions” that provide identification separate from the polynucleotide sequences used for forming homologies.

At806, it is determined if there are any additional timing indications. For example, the cell may be exposed to the timing indicator at a plurality of known times. Each of the plurality of known times may be recorded in a look-up table or other form for use in later correlating records of molecular events with time points.

At808, sequence data is obtained from the double-stranded polynucleotide. Sequencing, through any technique for DNA sequencing known to one of skill in the art, generates a sequence of the double-stranded polynucleotide including sequences introduced by the HDR template. The sequence data output from a polynucleotide sequencer is a computer file that is amenable to electronic analysis and manipulation.

In one implementation, all the DNA in the cell may be sequenced. In another implementation only the double-stranded polynucleotide may be sequenced. For example, if the double-stranded polynucleotide molecule is a vector, known primer sites on the vector may be used to sequence only the DNA of that vector and not the entirety of the DNA in the cell. In yet another implementation, polynucleotides in the region of the target site may be sequenced. For example, the target site in the double-stranded polynucleotide may be flanked with known sequences that can be used to design primers which specifically select and amplify the target site and any sequence integrated into the middle of the target site. Doing so captures any portions of HDR templates that were integrated into the target site.

At810, the molecular event is correlated with the known time. The sequence data obtained at808can be interpreted to identify a record of a molecular event and or a timing indicator. The interpretation may be as simple as identifying that the sequence of the middle portion of the HDR template is present in the sequence data. Recall that the HDR template is designed and intentionally inserted into a cell through transfection or another process. Thus, the sequence is known and can be used as a search query run against the content of the sequence data. Interpreting the meaning of finding such a sequence in the sequence data depends on the construction of the cell and association between a given signal and the HDR template. Thus, if the cell is designed so that detection of a signal (e.g., light) leads to incorporation of an HDR template with a middle portion having a sequence AGTTACGGA, then presence of the sequence AGTTACGGA in the sequence file serves as a record that the cell experienced light sufficient to trigger the relevant signaling pathway. The correlating may involve identifying in the sequence data a sequence from the second HDR template (i.e., molecular event) adjacent to a first sequence from the first HDR template (i.e., timing indication). In other words, the second HDR template is integrated into the double-stranded polynucleotide in the middle of the first HDR template that has been previously integrated. The sequences are “adjacent” if after integration into the double-stranded polynucleotide, nucleotides from one HDR template are contiguous with nucleotides from the other, or if there are only a small number of intervening nucleotides such as fewer than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nt.

Converting the identification of a sequence in sequence data to the identity of a signal and timing of the signal may be done by using a look-up table or other technique for correlating a DNA sequence with a type of signal and timing indicator. The correlation may be known based on the relationship between the engineered signaling pathway, a promoter affected by the engineered signaling pathway, and the sequence of the first and second HDR templates regulated by promoters. Reporting of the correlation and the timing may be performed, for example, by indicating the signal on a user interface of an electronic device such as a computer or a polynucleotide sequencer.

Illustrative Timing Techniques

FIG. 9shows a schematic900of a gene regulated by a genetic circuit creating an internal biological “clock” that results in creation of HDR templates at a timing driven by behavior of the genetic circuit. In addition to the manual timing described inFIG. 8, automatic timing based on biological clocks can be used to generate timing signals that are integrated into a polynucleotide.

There are many types of biological clocks both natural and synthetic. Natural autonomous cycles that may exist within a cell include the cell cycle, the metabolic cycle, photosynthesis activity, cycles caused by cell stress, etc. Natural cycles might come from neighboring cells such as electrical impulses from synapses. For cells that are part of a multi-cellular organism, there may be endocrine signals like cortisol (e.g., circadian rhythm), stress hormones, or development hormones (e.g., fruit ripening in plants). Any of these cycles may be harnessed to use periodic availability of particular molecules as timing signals that can regulate the ability of the cell to perform homology directed repair.

The cell cycle or cell-division cycle, for example, is the series of events that take place in a cell leading to its division and duplication of its DNA (DNA replication) to produce two daughter cells. In bacteria, which lack a cell nucleus, the cell cycle is divided into the B, C, and D periods. The B period extends from the end of cell division to the beginning of DNA replication. DNA replication occurs during the C period. The D period refers to the stage between the end of DNA replication and the splitting of the bacterial cell into two daughter cells. In cells with a nucleus, as in eukaryotes, the cell cycle is also divided into three periods: interphase, the mitotic (M) phase, and cytokinesis. During interphase, the cell grows, accumulating nutrients needed for mitosis, preparing it for cell division and duplicating its DNA. During the mitotic phase, the chromosomes separate. During the final stage, cytokinesis, the chromosomes and cytoplasm separate into two new daughter cells. To ensure the proper division of the cell, there are control mechanisms known as cell cycle checkpoints.

Two key classes of regulatory molecules, cyclins and cyclin-dependent kinases (CDKs), determine a cell's progress through the cell cycle. Cyclins form the regulatory subunits and CDKs the catalytic subunits of an activated heterodimer; cyclins have no catalytic activity and CDKs are inactive in the absence of a partner cyclin. When activated by a bound cyclin, CDKs perform a common biochemical reaction called phosphorylation that activates or inactivates target proteins to orchestrate coordinated entry into the next phase of the cell cycle. Different cyclin-CDK combinations determine the downstream proteins targeted. CDKs are constitutively expressed in cells whereas cyclins are synthesized at specific stages of the cell cycle, in response to various molecular signals.

Two families of genes, the cip/kip (CDK interacting protein/Kinase inhibitory protein) family and the INK4a/ARF (Inhibitor of Kinase 4/Alternative Reading Frame) family, prevent the progression of the cell cycle. Because these genes are instrumental in prevention of tumor formation, they are known as tumor suppressors.

The cip/kip family includes the genes p21, p27 and p57. They halt cell cycle in GI phase, by binding to, and inactivating, cyclin-CDK complexes. p21 is activated by p53 (which, in turn, is triggered by DNA damage e.g. due to radiation). p27 is activated by Transforming Growth Factor of β (TGF β), a growth inhibitor. The INK4a/ARF family includes p16INK4a, which binds to CDK4 and arrests the cell cycle in G1 phase, and p14ARF which prevents p53 degradation. Any of the regulatory molecules involved in the cell cycle may be used, for example by an engineered signaling pathway, to control an HDR template and associated homology directed repair behavior in order to create a genetic record that tracks the timing of the corresponding natural cellular cycle.

In addition to natural cycles or clocks, artificial cyclic events may be created by genetic circuits created through well-established synthetic biology techniques known to those of ordinary skill in the art. A synthetic genetic circuit(s) can be configured as a switch, a bi-stable switch, a toggle switch, an oscillator, a repressilator, a counter, an anticipator, a learner, a kill switch, a quorum sensor (sender or receiver), a two-way signaling system, and-gates, nor-gates, nand-gate, inverters, or-gates, engineered ecosystem circuits, single invertase memory modules, analog-to-digital converters, digital-to-analog converters or any combination thereof. A synthetic genetic circuit can be tunable and thus allow for modulation of expression of one or more reporter genes. In some embodiments, the synthetic genetic circuit can be designed using a program such as GenoCAD, Clotho framework, or j5. The synthetic genetic circuit can contain one or more reporter genes. Suitable reporter genes are generally known in the art. Such reporter genes include, but are not limited to, optically active proteins (e.g. green fluorescent protein and variants thereof (e.g. eGFP), red fluorescent protein and variants thereof (e.g. mCherry)), lacZ (produces beta-galatosidase), cat (produces chloramphenicol acetyltransferase), beta-lactamase, and other antibiotic resistance genes.

The reporter genes can be operatively coupled to one or more transcriptional control elements. As used herein, “transcriptional control element” can refer to any element of the synthetic genetic circuit, including proteins, DNA, RNA, or other molecules that can, either alone or in conjunction with other elements of the synthetic genetic circuit, stimulate and/or repress the transcription of one or more reporter genes within the synthetic genetic circuit. Such transcriptional control elements will be apparent to those in the art and include, but are not limited to operons and components thereof, bacterial repressors, eukaryotic promoters and elements therein, DNA binding proteins, signaling molecules, riboregulators, toe-hold switches, siRNA, and the like.

There are well-known gene networks that oscillate without the need for external chemical inducers. One oscillating synthetic genetic circuit that is a canonical feature within the field of synthetic biology is described in Stricker, J. et al.,A fast, robust and tunable synthetic gene oscillator,456 Nature 516 (2008) and Hasty, J., et al.,Synthetic Gene Network for Entraining and Amplifying Cellular Oscillations,88 Physical Review Letters 148101 (2002). A different network uses an orthogonal circuit containing negative feedback. This gene network causes elevated levels of a first signal protein only when levels of acyl-homoserine lactone (AHL) within the cell increased. However, when the target is further away, AHL input decreases, the circuit represses and concentration of signal protein attenuates. Voliotis, M., and Bowsher, C. G.,The magnitude and colour of noise in genetic negative feedback systems.40 Nucleic Acids Research 7084 (2012). Persons of ordinary skill in the art will understand how to construct these and other types of synthetic genetic circuits.

The illustrative gene oscillator902shown inFIG. 9is one nonlimiting example of a synthetic genetic circuit. The gene oscillator902includes a first operon with a promoter904that controls expression of a first transcription factor906and a first gene908. The first transcription factor906also up regulates the promoter904creating a positive feedback loop. Furthermore, the first transcription factor906also up regulates a second promoter910on a second operon. The second promoter910controls expression of a second transcription factor912and a second gene914. The second transcription factor912suppresses the first promoter904which in turn reduces expression of the first transcription factor906.

This arrangement creates oscillating behavior because initially the first transcription factor906will cause the first promoter904to create ever larger amounts of the first transcription factor906leading to increase in expression of the second gene914controlled by the second promoter910. The second gene914is responsible for increasing homologous directed repair using an HDR template916. However, increasing concentration of the first transcription factor906also leads to increased expression of the second transcription factor912. Because the second transcription factor912suppresses activity of the first promoter904, it will eventually lead to decreased expression of the second gene914. Thus, the availability of the HDR template916will fluctuate with a known periodicity based on the behavior of the gene oscillator902.

As described above, there are multiple ways in which increased integration of an HDR template may be regulated. For example, the second gene914may directly code for the HDR template916as RNA or as RNA that is later transcribed into DNA. Alternatively, the second gene914may be involved in increasing the number of DSBs into which the HDR template916may be integrated. This can be done by increasing the number of enzymes configured to create DSBs in a double-stranded polynucleotide. Thus, the second gene914may encode the protein that functions as a nuclease or may include another gene product that assists with the functioning of the nuclease such as, for example a gRNA that includes a protospacer element that guides Cas9 to a particular target site by hybridizing with one strand of the target site. The HDR template916may include features similar to any of the other HDR templates described in this disclosure. Incorporation of this HDR template916into a double-stranded polynucleotide creates a record of a timing indicator that occurs at a periodicity established by the design of the gene oscillator902.

FIG. 10shows a diagram1000of a timing indicator1002controlling behavior of an operon1004. The timing indicator1002may be based on a manually pulsed signal as described in conjunction withFIG. 8or based on a natural biological cycle or a synthetic genetic circuit as described conjunction withFIG. 9. The operon1004can include a promoter1006, an operator1008, and a gene1010. The gene encodes RNA1012, the expression of which is up regulated at a frequency influenced by the timing indicator1002.

The RNA1012may be translated into an enzyme1014that functions as a site-specific nuclease which creates DSBs at specific locations in double-stranded polynucleotides. Alternatively, the RNA1012may be gRNA1016that guides a Cas9 nuclease to a specific target site. Additionally, the RNA1012may be an HDR template1018or may be transcribed into ssDNA that functions as the HDR template1018. Thus, through any of the mechanisms described above up regulation of the gene1010leads to increased integration of HDR templates into a specific target site on a double-stranded polynucleotide. This increase is affected either by increasing the enzymes that create the DSBs which are repaired through homology directed repair, guiding existing enzymes to specific locations in order to create the DSBs, or increasing copies of the HDR templates themselves.

A double-stranded polynucleotide1020, which may be any of the double-stranded polynucleotides discussed elsewhere in this disclosure, includes a target site1022with a cut site1024. Homology between the ends of the HDR template1018and the target site1022enable homology directed repair of a DSB created at the cut site1024as shown inFIGS. 1 and 2. This results in integration of a middle portion1026of the HDR template1018into the target site1022of the double-stranded polynucleotide1020. Presence of the nucleotide sequence corresponding to this middle portion1026provides a genetic record of the timing indicator1002. The middle portion1026of the HDR template1018can include a further cut site1028. A DSB may be formed in this cut site1028by a different enzyme and the DSB may be repaired by a different HDR template. Thus, each cycle of the timing indicator1002creates an opportunity for integration of an HDR template configured for insertion into the middle portion1026of the HDR template1018. Alternatively, the HDR template1018may be configured for insertion into the cut site1028included in its own middle portion1026. This will lead to iterative insertion of the HDR template1018with each cycle of the timing indicator1002. This configuration of a cell can create a record of time by recording the number of timing indicators1002that occurred. For example, five insertions of the HDR template1018indicates that five cycles of the timing indicator1002have elapsed. Alternatively, HDR templates corresponding to molecular events may also be integrated into the double-stranded polynucleotide1020and the presence of the HDR template1018corresponding to the timing indicator1002may provide an indication of the frequency and temporal spacing of the molecular events.

FIG. 11shows a diagram1100illustrating insertion of an HDR template caused by molecular event1102following insertion of an HDR template in response to a timing indicator1104. In this example, integration of a first HDR template1106caused by the timing indicator1104creates a location in the double-stranded polynucleotide1108for insertion of a second HDR template1110caused by the molecular event1102. The molecular event1102may be the same or similar to any of the types of molecular events described previously in this disclosure. The timing indicator1104may be the same or similar to any of the other timing indicators described in this disclosure.

The timing indicator1104leads to creation of the first HDR template1106or to creation of enzymes that facilitate integration of the first HDR template1106into the double-stranded polynucleotide1108. The first HDR template1106includes homology regions1112that are homologous to a target site1114on the double-stranded polynucleotide1108. Thus, when a DSB is formed in the target site1114, the first HDR template1106is able to repair the DSB through homology directed repair. This repair introduces the sequence in the middle of the first HDR template1106into the sequence of the double-stranded polynucleotide1108. The first HDR template1106includes a middle section1116that can form homologies with portions of the second HDR template1110. This middle section1116of the first HDR template1106may also include a cut site1118configured to be cut by a nuclease. The first HDR template1106includes an identifier region1120that is not homologous to the target site1114or to any portion of the first HDR template1106. This identifier region1120may also be unique in that this sequence is not the same as any portion of the double-stranded polynucleotide1108or any portion of the first HDR template1106. Thus, incorporation of the identifier region1120in the double-stranded polynucleotide1108provides an indication of an occurrence of the timing indicator1104that can be uniquely identified and that will be retained even following further iterative insertions at the cut site1118introduced by the first HDR template1106.

The molecular event1102leads to creation of the second HDR template1110or to changes (e.g., increase in number or activity of nucleases) that increase the ability of already existing HDR templates1110to be incorporated into the double-stranded polynucleotide1108. The second HDR template1110includes homology regions1122that are homologous to the middle portion1116of the first HDR template1106. This allows insertion of the first HDR template1106to provide a location for subsequent insertion of the second HDR template1110. Insertion of the second HDR template1110introduces into the double-stranded polynucleotide1108the portions of the second HDR template1110that are flanked by the homologous regions1122. This includes a middle portion1124that may be homologous to the homologous portions1112of the first HDR template1106. The middle portion1124can also include a cut site1126. This cut site1126may be the same as the cut site present in the target site1114of the double-stranded polynucleotide1108. Thus, the second HDR template1110may include a portion that introduces the same target site1114originally present in the double-stranded polynucleotide1108. This makes it so that after integration of the second HDR template1110, the double-stranded polynucleotide1108is once again capable of incorporating the first HDR template1106the next time a timing indicator1104occurs. Furthermore, the second HDR template1110may include its own identifier region1128. Similar to the identifier region1120in the first HDR template1106, this identifier region1128may be both unique and lacking homologies in either the second HDR template1106or the double-stranded polynucleotide1108. Thus, each instance of the identifier region1128in the double-stranded polynucleotide1108provides a record showing that the molecular event1102occurred.

Because the first HDR template1106introduces a target site1116that is capable of incorporating the second HDR template1110and the second HDR template1110introduces a target site1124which is capable of incorporating the first HDR template1106, this leads to alternative integration of the two HDR templates1106,1110. In this implementation, each time the timing indicator1104occurs, the molecular event1102may be logged. For example, if the timing indicator1104occurs with the frequency of about 24 hours, and the molecular event1102is temperature, then the temperature sensed by the cell may be recorded every 24 hours. Different temperatures may be recorded by having multiple different HDR templates that each respond to engineered signaling pathways triggered by different temperature ranges. Each of these various HDR templates may include the same homologous portions1102and same middle portion1124so that the ability to have alternative integration with the first HDR template1106is not altered. However, the identifier region1128may be different for each temperature range in order to create a nucleotide log that records changes in temperature.

FIG. 12shows a diagram1200illustrating insertion of an HDR template caused by a timing indicator1202following insertion of an HDR template in response to a molecular event1204. In this example, integration of a first HDR template1206caused by the molecular event1204creates a location in the double-stranded polynucleotide1208for insertion of a second HDR template1210caused by the timing indicator1202. Diagram1200inFIG. 12is similar to diagram1100inFIG. 11but shows a different sequence of the HDR template integration. Here, integration of the first HDR template1206in response to the molecular event1204occurs first and creates a target site for insertion of the second HDR template1210triggered by the timing indicator1202.

The first HDR template1206includes homologous regions1212that form homologies with a target site1214on the double-stranded polynucleotide1208. Like the HDR templates described inFIG. 11, the first HDR template1206also includes a middle region1216, a cut site1218, and an identifier region1220. Integration of the first HDR template1206into the double-stranded polynucleotide1208creates a target site into which the second HDR template1210may be inserted based on the occurrence of the timing indicator1202.

The second HDR template1210also includes homologous regions1222, a middle region1224, a cut site1226, and an identifier region1228. The homologous regions1222are homologous to the middle region1216of the first HDR template1206. And, the middle region1224of the second HDR template introduces a target site into the double-stranded polynucleotide1208for insertion of the first HDR template1206if made available due to a molecular event1204. Thus, in this implementation each time an event is sensed and this corresponding HDR template1206is integrated into the double-stranded polynucleotide1208, it is possible to make a log of the time as represented by the timing indicator1202with the identification region1228included in the second HDR template1210. This identification region1228may be slightly different in different versions of the second HDR template1204based on triggering by timing indicators1202that occur at different times. Thus, the specific sequence of the identification region1228varies based on which version of the second HDR template1210is abundant and that varies based on time. Thus, after recording of each molecular event1204it is possible to record a time which will be approximately the time that the molecular event occurred.

FIG. 13shows a diagram1300of integration of polynucleotide sequences representing timing indicators in a double-stranded polynucleotide1302that is continuously logging a molecular event.FIG. 11andFIG. 12show two different implementations in which alternating copies of HDR templates caused by molecular events and caused by timing indicators may be incorporated into a double-stranded polynucleotide. The diagram1300illustrates a similar architecture but shows how sequential and iterative logging of molecular events can be combined with logging of time points. In one implementation, a cell may be continuously logging a molecular event such as temperature, pH, presence of a chemical, salinity, etc. through techniques discussed earlier in this disclosure. Later evaluation of such a log may provide a record of how the environmental conditions of the cell changed and the order in which different conditions were experienced, but will not provide a ready way to correlate that with the times at which such conditions were experienced by the cell.

Occurrence of the molecular event may trigger creation and or introduction of a first HDR template1304into a double-stranded polynucleotide1302. Multiple copies of the first HDR template1304may be created each time the event is sensed or the amount of copies may be proportional to the strength of the signal corresponding to the event. The double-stranded polynucleotide1302may include a target site in a cut site as described earlier. The first HDR template1304triggered in response to the molecular event can include an identifier region1306. The first HDR template1304may be structured so that integration of one copy of the HDR template as a target site for subsequent, iterative insertion of the HDR template1304.

The double-stranded polynucleotide following integration of multiple copies of the first HDR template1304is represented as a schematic1308. This schematic1308shows how the nucleotide sequence of the double-stranded polynucleotide includes multiple copies of the identifier region1306. This iterative insertion of the first HDR template1304may continue so long as the signal caused by the molecular event is present.

A timing indicator operating on a periodic or non-periodic but known frequency can cause a second HDR template1310to be present and/or available for integration into the double-stranded polynucleotide at a frequency corresponding to the timing indicator. This second HDR template1310can include an identifier region1312that is different from the identifier region1306present in the first HDR template1304associated with the molecular event. The second HDR template1310may have the same 3′-end, 5′-end, and middle portions as the first HDR template1304. This allows the second HDR template1310to be integrated into a portion of the double-stranded polynucleotide1302created by repair of a DSB with the first HDR template1304.

Schematic1314shows a structure of the double-stranded polynucleotide after the first HDR template1304has been inserted three times and the second HDR template1310has been inserted once. Schematic1316shows a structure of the double-stranded polynucleotide in somewhat simplified form after three cycles of the timing indicator have resulted in incorporation of the second HDR template1310three different times. Temporal patterns of the logging of the molecular event become evident when the identifier region1312of the second HDR template1310is present in the double-stranded polynucleotide. In this example, the schematic1316shows an increasing frequency of integration of the first HDR template1304. Prior to the first instance of the identifier region1312caused by the timing indicator, three copies of the first HDR template1304are added. After the first identifier region1312, four copies of the first HDR template1304are added before the next timing indicator occurs. Following the second incorporation of the identifier region1312and prior to the third, five copies of the first HDR template1304are added to the double-stranded polynucleotide. If the timing indicator occurs on a periodic schedule, then the frequency of the molecular event is increasing over time or, depending on the relationship between the engineered signaling pathway and the first HDR template1304, the strength of the signal for the molecular event may be increasing over time.

One having ordinary skill in the art will appreciate that variations on this pattern can indicate different temporal relationships between behavior of the molecular event and the timing indicator. Thus, by creating a system in which both HDR templates corresponding to the sensed event and HDR templates corresponding to the timing indicator can be integrated into the same double-stranded polynucleotide, increased information about the timing of the molecular event is available from the double-stranded polynucleotide.

Illustrative System and Computing Devices

FIG. 14shows an illustrative architecture1400for implementing and interacting with DNA molecules recording logs and timing of molecular events by use of HDR and timing indicators as described above. The architecture may include any of a digital computer1402, an oligonucleotide synthesizer1404, an automated system1406, and/or a polynucleotide sequencer1408. The architecture1400may also include other components besides those discussed herein.

As used herein. “digital computer” means a computing device including at least one hardware microprocessor1410and memory1412capable of storing information in a binary format. The digital computer1402may be a supercomputer, a server, a desktop computer, a notebook computer, a tablet computer, a game console, a mobile computer, a smartphone, or the like. The hardware microprocessor1410may be implemented in any suitable type of processor such as a single core processor, a multicore processor, a central processing unit (CPU), a graphical processing unit (GPU), or the like. The memory1412may include removable storage, non-removable storage, local storage, and/or remote storage to provide storage of computer readable instructions, data structures, program modules, and other data. The memory1412may be implemented as computer-readable media Computer-readable media includes, at least, two types of media, namely computer-readable storage media and communications media. Computer-readable storage media includes volatile and non-volatile, removable and non-removable media implemented in any method or technology for storage of information such as computer-readable instructions, data structures, program modules, or other data. Computer-readable storage media includes, but is not limited to, RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other non-transmission medium that can be used to store information for access by a computing device.

In contrast, communications media may embody computer readable instructions, data structures, program modules, or other data in a modulated data signal, such as a carrier wave, or other transmission mechanism. As defined herein, computer-readable storage media and communications media are mutually exclusive.

The digital computer1402may also include one or more input/output devices(s)1414such as a keyboard, a pointing device, a touchscreen, a microphone, a camera, a display, a speaker, a printer, and the like.

An HDR template designer1416may be included as part of the digital computer1402, for example, as instructions stored in the memory1412. The HDR template designer1416may design HDR templates based on sequences of target sites, sequences of dsDNA molecules, enzyme recognition sites, etc. In one implementation, the HDR template designer1416may design HDR templates to avoid cross talk between different signal recording pathways. The HDR template designer1416may also compare percent similarity and hybridization conditions for potential HDR templates as well as portions of the HDR templates. For example, the HDR template designer1416may design HDR templates to avoid the formation of hairpins as well as to prevent or minimize annealing between HDR templates. The HDR template designer1416may also design HDR templates to maximize a difference between the 3′-end sequence, 5′-end sequence, and/or middle sequence. For example, the difference may be G:C content and the HDR template designer1416may design sequences with a preference for increasing the G:C content difference between the end sequences and the middle sequence.

The digital computer1402may also include a look-up table1418. However, the look-up table1418may be part of a hardware device that is physically separate from the digital computer1402. The look-up table1418includes the correspondence between the sequence of an HDR template and a signal or a time point. For example, the information that expression of a given HDR template is up regulated in the presence of a given signal is one example of a correspondence that may be stored in the look-up table1418. Users may make entries into the look-up table1418that indicate the times a given timing indicator was manually pulsed. The look-up table1418may store any number of different associations between signals/timing indicators and HDR templates. The look-up table1418may be pre-calculated and stored in static program storage, calculated (or “pre-fetched”) as part of a program's initialization phase (e.g., memorization), or even stored in hardware in an application-specific platform.

A sequence data analyzer1420may analyze sequence data1422generated by the polynucleotide sequencer1408. The sequence data analyzer1420may be implemented as instructions stored in the memory1412. Thus, sequence data1422may be provided to the sequence data analyzer1420which analyzes the sequence data1422at least in part by comparison to nucleotide sequences contained in the look-up table1418. The sequence data analyzer1420may identify which signals were detected by a cell1424and may identify timing indicators included in the DNA of the cell1424. Depending on the design of the cell1424, the sequence data analyzer1420may also identify a signal strength, relative signal strength, order of different signals, signal duration, timing of signals, or other characteristic of one or more signals represented in the sequence data1422. As used herein, “cell” includes biological cells, minimal cells, artificial cells, and synthetic cells. A detectable molecular event is recognized by the cell, and the cell responds by modifying its genetic material.

Information about timing indicators and nucleotide sequences may be correlated with “wall-clock” time or the timing of a clock in the digital computer1402by a correlator of timing indications1426. This correlator of timing indications1426may reference information from the look-up table1418. For example, the sequence data1422may be searched to identify a polynucleotide sequence identified in the look-up table1418as corresponding to a genetic oscillator that has periodicity of 2.5 to 2.7 hours. Then, the correlator of time indications1426can use a known start time to derive a range of wall-clock times for various insertions of HDR templates generated by the timing of the genetic oscillator. Because the periodicity of the genetic oscillator is approximate, the range of possible values for wall-clock time will increase as the number of timing cycles increases. The correlator of timing indications1426can account for this range of possible times and provide estimated wall-clock times or a range of possible wall-clock times for various molecular events logged by the cell1424.

In order to manipulate the DNA and potentially RNA that makes up the HDR templates and dsDNA, the digital computer1402may communicate with other devices through one or more I/O data interfaces1428. The I/O data interface(s)1428can exchange instructions and data with other devices such as the oligonucleotide synthesizer1404, the automated system1406, and the polynucleotide sequencer1408.

The oligonucleotide synthesizer1404chemically synthesizes oligonucleotides based on instructions received as electronic data. The synthesized oligonucleotides may be used as HDR templates, as dsDNA molecules that provide target sites, as plasmids, vectors, or other components. Thus, in some implementations, the sequence of nucleotides which is provided to the oligonucleotide synthesizer1404may come from the HDR template designer1416.

A number of methods for DNA synthesis and commercial oligonucleotide synthesizers are available. Methods for DNA synthesis include solid-phase phosphoramidite synthesis, microchip-based oligonucleotide synthesis, ligation-mediated assembly, polymerases chain reaction PCR-mediated assembly, and the like. For example, such synthesis can be performed using an ABI 394 DNA Synthesizer (Applied Biosystems, Foster City, Calif.). One having ordinary skill in the art will understand how to use an oligonucleotide synthesizer to generate an oligonucleotide with a desired sequence.

The term “oligonucleotide” as used herein is defined as a molecule including two or more nucleotides. Oligonucleotides include probes and primers. Oligonucleotides used as probes or primers may also include nucleotide analogues such as phosphorothioates, alkylphosphorothioates, peptide nucleic acids, or intercalating agents. The introduction of these modifications may be advantageous in order to positively influence characteristics such as hybridization kinetics, reversibility of the hybrid-formation, stability of the oligonucleotide molecules, and the like.

The automated system1406may include any type of robotics, automation, or other system for automating one or more manipulations that may be performed on the dsDNA with the enzymes and/or the HDR templates. The automated system1406may be used in conjunction with manual operations such that the totality of operations needed to be performed to practice the techniques of this disclosure are done so in a hybrid manner in which some are performed by the automated system1406and others manually.

In one implementation, the automated system1406may include a microfluidics system. An illustrative microfluidics system may be configured to move small volumes of liquid according to techniques well-understood by those of ordinary skill in the art. As used herein, the automated system1406may include other equipment for manipulating DNA beyond that expressly shown inFIG. 14such as, for example, a thermocycler.

The automated system1406may include a cell-free system that can be implemented in part by microfluidics. The cell-free system may also be implemented as an artificial cell or a minimal cell. As used herein the term “cell” encompasses natural cells, artificial cells, and minimal cells unless context clearly indicates otherwise. The automated system1406may include one or more natural cells such as a cell in culture. A culture of cells in the automated system1406may be manipulated by an automated cell culture system. An artificial cell or minimal cell is an engineered particle that mimics one or many functions of a biological cell. Artificial cells are biological or polymeric membranes which enclose biologically active materials. As such, nanoparticles, liposomes, polymersomes, microcapsules, detergent micelles, and a number of other particles may be considered artificial cells. Micro-encapsulation allows for metabolism within the membrane, exchange of small molecules and prevention of passage of large substances across it. Membranes for artificial cells can be made of simple polymers, crosslinked proteins, lipid membranes or polymer-lipid complexes. Further, membranes can be engineered to present surface proteins such as albumin, antigens, Na/K-ATPase carriers, or pores such as ion channels. Commonly used materials for the production of membranes include hydrogel polymers such as alginate, cellulose and thermoplastic polymers such as hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA), poly-acrylonitrile-polyvinyl chloride (PAN-PVC), as well as variations of the above-mentioned materials.

Minimal cells, also known as proto-cells, are cells that help all the minimum requirements for life. Minimal cells may be created by a top-down approach that knocks out genes in a single-celled organism until a minimal set of genes necessary for life are identified.Mycoplasma mycoides, E. coli, andSaccharomyces cerevisiae, are examples of organisms that may be modified to create minimal cells. One of ordinary skill in the art will recognize multiple techniques for generating minimal cells.

The cell-free system includes components for DNA replication and repair such as nucleotides, DNA polymerase, and DNA ligase. The cell-free system will also include dsDNA that includes at least one initial target site for creating a DSB. The dsDNA may be present in the vector that includes one or more operons. The cell-free system will also include buffers to maintain pH and ion availability. Furthermore, the cell-free system may also include the enzymes used for creating DSBs in dsDNA and the HDR templates used for repairing dsDNA. Some cell-free systems may include genes encoding the enzymes and HDR templates. To prevent enzymes from remaining when their respective cutting functions are no longer desired, the cell-free system may include proteolytic enzymes that specifically break down nucleases.

In a cell-free system, particular components may be added when needed either by moving volumes of liquid together with microfluidics or by increasing the expression of gene products that leads to synthesis of enzymes, HDR templates, etc.

The automated system1406may include a structure, such as at least one chamber, which holds one or more DNA molecules. The chamber may be implemented as any type of mechanical, biological, or chemical arrangement which holds a volume of liquid, including DNA, to a physical location. For example, a single flat surface having a droplet present thereon, with the droplet held by surface tension of the liquid, even though not fully enclosed within a container, is one implementation of a chamber.

The automated system1406may perform many types of manipulations on DNA molecules. For example, the automated system1406may be configured to move a volume of liquid from one chamber to another chamber in response to a series of instructions from the I/O data interface1428.

The polynucleotide sequencer1408may sequence DNA molecules using any technique for sequencing polynucleotides known to those skilled in the art including classic dideoxy sequencing reactions (Sanger method), sequencing by synthesis using reversibly terminated labeled nucleotides, pyrosequencing, nanopore sequencing, SOLiD sequencing, chemical-sensitive field effect transistor (chemFET) sequencing, and ion semiconductor sequencing. The polynucleotide sequencer1408may be configured to sequence all or part of a dsDNA molecule modified according to any of the techniques described above and provide the sequence data1422to the digital computer1402.

A cell1424may be prepared for sequencing by extracting nucleic acids according to standard methods in the art. For example, DNA from a cell can be isolated using various lytic enzymes, chemical solutions, or extracted by nucleic acid binding resins following instructions provided by a manufacturer. DNA contained in extracted sample may be detected by amplification procedures such as PCR or hybridization assays according to methods widely known in the art.

The sequence data1422generated by sequencing can be sent from the polynucleotide sequencer1408to the digital computer1402for analysis by the sequence data analyzer1420, the correlator of timing indications1426, and also for presentation on an output device1414.

Restriction enzymes (restriction endonucleases) are present in many species and are capable of sequence-specific binding to DNA (at a target or recognition site), and cleaving DNA at or near the site of binding. Over 3000 restriction enzymes have been studied in detail, and more than 600 of these are available commercially. Naturally occurring restriction endonucleases are categorized into four groups (Types I, II III, and IV) based on their composition and enzyme cofactor requirements, the nature of their target site, and the position of their DNA cleavage site relative to the target site. All types of enzymes recognize specific short DNA sequences and carry out the endonucleolytic cleavage of DNA to give specific fragments with terminal 5′-phosphates. One type of restriction enzyme, Type II enzymes, cleave within or at short specific distances from a recognition site; most require magnesium; single function (restriction) enzymes independent of methylase. Type II enzymes form homodimers, with recognition sites that are usually undivided and palindromic and 4-8 nucleotides in length. They recognize and cleave DNA at the same site, and they do not use ATP or AdoMet for their activity—they usually require only Mg2+as a cofactor. Common type II restriction enzymes include HhaI, HindIII, NotI, EcoRI, and BglI. Restriction enzymes may cut dsDNA in a way that leaves either blunt ends or sticky ends. Protocols for creating a DSB in dsDNA with restriction enzymes are well known to those skilled in the art. Restriction digest is a common molecular biology technique and is typically performed using the reagents and protocols provided in a commercially available restriction digest kit. Examples of companies that provide restriction digest kits include New England BioLabs, Promega, Sigma-Aldrich, and Thermo Fisher Scientific. Each of these companies provides restriction digest protocols on their website.

Homing endonucleases (HEs), which are also known as meganucleases, are a collection of double-stranded DNases that have large, asymmetric recognition sites (12-40 nt) and coding sequences that are usually embedded in either introns or inteins. Introns are spliced out of precursor RNAs, while inteins are spliced out of precursor proteins. They catalyze the hydrolysis of genomic DNA within the cells that synthesize them, but do so at few, or even a single, location(s) per genome. HE recognition sites are extremely rare. For example, an 18 nt recognition sequence will occur only once in every 7×1010nucleotides of random sequence. This is equivalent to only one site in 20 mammalian-sized genomes. However, unlike restriction endonucleases, HEs tolerate some sequence degeneracy within their recognition sequence. Thus, single base changes do not abolish cleavage but reduce its efficiency to variable extents. As a result, their observed sequence specificity is typically in the range of 10-12 nt. Examples of suitable protocols using HEs may be found in Flick, K. et al.,DNA Binding in Cleavage by the Nuclear Introns-Encoded Homing Endonuclease I-Ppol,394 Nature 96 (1998) and Chevalier, B. et al.,Design, Activity. and Structure of a Highly Specific Artificial Endonuclease,10 Molecular Cell 895 (2002).

Zinc finger nucleases (ZFNs) are synthetic proteins consisting of an engineered zinc finger DNA-binding domain fused to the cleavage domain of the FokI restriction endonuclease. ZFNs can be used to induce DSBs in specific DNA sequences and thereby promote site-specific homologous recombination and targeted manipulation of genomic loci in a variety of different cell types. The introduction of a DSB into dsDNA may enhance the efficiency of recombination with an exogenously introduced HDR template. ZFNs consist of a DNA-binding zinc finger domain (composed of three to six fingers) covalently linked to the non-specific DNA cleavage domain of the bacterial FokI restriction endonuclease. ZFNs can bind as dimers to their target DNA sites, with each monomer using its zinc finger domain to recognize a half-site. Dimerization of ZFNs is mediated by the FokI cleavage domain which cleaves within a five or six nucleotide “spacer” sequence that separates the two inverted “half sites.” Because the DNA-binding specificities of zinc finger domains can in principle be re-engineered using one of various methods, customized ZFNs can be constructed to target nearly any DNA sequence. One of ordinary skill in the art will know how to design and use ZFNs to create DSBs in dsDNA at a desired target site. Some suitable protocols are available in Philipsbom, A. et al.,Microcontact printing of axon guidance molecules for generation of graded patterns,1 Nature Protocols 1322 (2006); John Young and Richard Harland,Targeted Gene Disruption with Engineered Zinc Finger Nucleases(ZFNs), 917XenopusProtocols 129 (2012), and Hansen, K. et al.Genome Editing with CompoZr Custom Zinc Finger Nucleases(ZFNs), 64 J. Vis. Exp. 3304 (2012).

TALENs are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (i.e., a nuclease which cuts DNA strands). Transcription activator-like effectors (TALEs) can be engineered to bind practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations. The restriction enzymes can be introduced into cells, for use in gene editing or for genome editing in situ. The DNA binding domain contains a repeated highly conserved 33-34 amino acid sequence with divergent 12thand 13thamino acids. These two positions, referred to as the Repeat Variable Diresidue (RVD), are highly variable and show a strong correlation with specific nucleotide recognition. This straightforward relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA-binding domains by selecting a combination of repeat segments containing the appropriate RVDs. Notably, slight changes in the RVD and the incorporation of “nonconventional” RVD sequences can improve targeting specificity. One of ordinary skill in the art will know how to design and use TALENs to create DSBs in dsDNA at a desired target site. Some suitable protocols are available in Hermann. M. et al.,Mouse Genome Engineering Using Designer Nucleases,86 J. Vis. Exp. 50930 (2014) and Sakuma, T. et al.,Efficient TALEN Construction and Evaluation Methods for Human Cell and Animal Applications,18(4) Genes Cells 315 (2013).

In the CRISPR/Cas nuclease system, the CRISPR locus, encodes RNA components of the system, and the Cas (CRISPR-associated) locus, encodes proteins. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated polynucleotide cleavage.

The Type II CRISPR is one of the most well characterized systems and carries out targeted double-stranded breaks in four sequential steps. First, two non-coding RNAs, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition. In engineered CRISPR/Cas9 systems, gRNA also called single-guide RNA (“sgRNA”) may replace crRNA and tracrRNA with a single RNA construct that includes the protospacer element and a linker loop sequence. Standard Watson-Crick base-pairing includes: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C). In addition, it is also known in the art that for hybridization between two RNA molecules (e.g., dsRNA), guanine (G) base pairs with uracil (U). In the context of this disclosure, a guanine (G) is considered complementary to a uracil (U), and vice versa. As such, when a G/U base-pair can be made at a given nucleotide position a protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule, the position is not considered to be non-complementary, but is instead considered to be complementary. Use of gRNA may simplify the components needed to use CRISPR/Cas9 for genome editing. The Cas9 species of different organisms have different PAM sequences. For example,Streptococcus pyogenes(Sp) has a PAM sequence of 5′-NGG-3′, Staphylococcus aureus(Sa) has a PAM sequence of 5′-NGRRT-3′ or 5′-NGRRN-3′, Neisseria meningitidis(NM) has a PAM sequence of 5′-NNNNGATT-3′, Streptococcus thermophilus(St) has a PAM sequence of 5′-NNAGAAW-3′, Treponema denticola(Td) has a PAM sequence of 5′-NAAAAC-3′.

Finally, Cas9 mediates cleavage of target DNA to create a DSB within the protospacer. Activity of the CRISPR/Cas system in nature comprises three steps: (i) insertion of alien DNA sequences into the CRISPR array to prevent future attacks, in a process called ‘adaptation,’ (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference with the alien polynucleotide. The alien polynucleotides come from viruses attaching the bacterial cell. Thus, in the bacterial cell, several of the so-called ‘Cas’ proteins are involved with the natural function of the CRISPR/Cas system and serve roles in functions such as insertion of the alien DNA, etc.

CRISPR may also function with nucleases other than Cas9. Two genes from the Cpf1 family contain a RuvC-like endonuclease domain, but they lack Cas9's second HNH endonuclease domain. Cpf1 cleaves DNA in a staggered pattern and requires only one RNA rather than the two (tracrRNA and crRNA) needed by Cas9 for cleavage. Cpf1's preferred PAM is 5′-TTN, differing from that of Cas9 (3′-NGG) in both genomic location and GC-content. Mature crRNAs for Cpf1-mediated cleavage are 42-44 nucleotides in length, about the same size as Cas9's, but with the direct repeat preceding the spacer rather than following it. The Cpf1 crRNA is also much simpler in structure than Cas9's; only a short stem-loop structure in the direct repeat region is necessary for cleavage of a target. Cpf1 also does not require an additional tracrRNA. Whereas Cas9 generates blunt ends 3 nt upstream of the PAM site, Cpf1 cleaves in a staggered fashion, creating a five nucleotide 5′ overhang 18-23 nt away from the PAM.

Other CRISPR-associated proteins besides Cas9 may be used instead of Cas9. For example, CRISPR-associated protein 1 (Cas1) is one of the two universally conserved proteins found in the CRISPR prokaryotic immune defense system. Cas1 is a metal-dependent DNA-specific endonuclease that produces double-stranded DNA fragments. Cas1 forms a stable complex with the other universally conserved CRISPR-associated protein, Cas2, which is part of spacer acquisition for CRISPR systems.

There are also CRISPR/Cas9 variants that do not use a PAM sequence such as NgAgo. NgAgo functions with a 24-nucleotide ssDNA guide and is believed to cut 8-11 nucleotides from the start of this sequence. The ssDNA is loaded as the protein folds and cannot be swapped to a different guide unless the temperature is increased to non-physiological 55 C. A few nucleotides in the target DNA are removed near the cut site. Techniques for using NgAgo are described in Gao, F. et al.,DNA-guided Genome Editing Using the Natronobacterium Gregoryi Argonaute,34 Nature Biotechnology 768 (2016).

DSBs may be formed by making two single-stranded breaks at different locations creating a cut DNA molecule with sticky ends. Single-strand breaks or “nicks” may be formed by modified versions of the Cas9 enzyme containing only one active catalytic domain (called “Cas9 nickase”). Cas9 nickases still bind DNA based on gRNA specificity, but nickases are only capable of cutting one of the DNA strands. Two nickases targeting opposite strands are required to generate a DSB within the target DNA (often referred to as a “double nick” or “dual nickase” CRISPR system). This requirement dramatically increases target specificity, since it is unlikely that two off-target nicks will be generated within close enough proximity to cause a DSB. Techniques for using a dual nickase CRISPR system to create a DSB are described in Ran, et al.,Double Nicking by RNA-Guided CRISPR Cas9for Enhanced Genome Editing Specificity,154 Cell 6:1380 (2013).

In certain embodiments, any of the enzymes described in this disclosure may be a “functional derivative” of a naturally occurring protein. A “functional derivative” of a native sequence polypeptide is a compound having a qualitative biological property in common with a native sequence polypeptide. “Functional derivatives” include, but are not limited to, fragments of a native sequence and derivatives of a native sequence polypeptide and its fragments, provided that they have a biological activity in common with a corresponding native sequence polypeptide. A biological activity contemplated herein is the ability of the functional derivative to hydrolyze a DNA substrate into fragments. The term “derivative” encompasses both amino acid sequence variants of polypeptide, covalent modifications, and fusions thereof. Suitable derivatives of an enzyme or a fragment thereof include but are not limited to mutants, fusions, covalent modifications of the protein or a fragment thereof. The enzyme, or a fragment thereof, as well as derivatives or a fragment thereof, may be obtainable from a cell or synthesized chemically or by a combination of these two procedures. The cell may be a cell that naturally produces the enzyme. A cell that naturally produces enzyme may also be genetically engineered to produce the endogenous enzyme at a higher expression level or to produce the enzyme from an exogenously introduced polynucleotide, which polynucleotide encodes an enzyme that is the same or different from the endogenous enzyme. In some cases, a cell does not naturally produce the enzyme and is genetically engineered to produce the enzyme. The engineering may include adding the polynucleotide encoding the enzyme under the control of a promoter. The promoter may be an inducible promoter that is activated in response to a signal. The promoter may also be blocked by a different signal or molecule.

Illustrative Embodiments

The following clauses described multiple possible embodiments for implementing the features described in this disclosure. The various embodiments described herein are not limiting nor is every feature from any given embodiment required to be present in another embodiment. Any two or more of the embodiments may be combined together unless context clearly indicates otherwise. As used herein in this document, “or” means and/or. For example, “A or B” means A without B, B without A, or A and B. As used herein, “comprising” means including all listed features and potentially including addition of other features that are not listed. “Consisting essentially of” means including the listed features and those additional features that do not materially affect the basic and novel characteristics of the listed features. “Consisting of” means only the listed features to the exclusion of any feature not listed.

Clause 1. A system for creating a temporal record in a polynucleotide log, the system comprising: a gene oscillator that creates a signal at a periodicity; a double-stranded polynucleotide having a target site; an enzyme configured to create a double strand break (DSB) in the double-stranded polynucleotide at a cut site in the target site; and a gene encoding a homology directed repair (HDR) template configured for insertion into the cut site, the HDR template including a middle portion that is not homologous to the target site, the gene expressing the HDR template based on presence of the signal relative to a threshold level, wherein the HDR template is incorporated into the double-stranded polynucleotide with a frequency that is based on the periodicity.

Clause 2. The system of clause 1, wherein the system comprises a single eukaryotic cell or a single prokaryotic cell.

Clause 3. The system of clause 1 or 2, wherein the enzyme comprises CRISPR/Cas and a gRNA having a protospacer element that hybridizes with one strand of the target site.

Clause 4. The system of any of clauses 1-3, wherein the middle portion comprises a second cut site and the system further comprises a second enzyme configured to create a DSB at the second cut site and a second gene encoding a second HDR configured for insertion into the second cut site, the second gene expressing the HDR template in response to a second signal generated by a molecular event.

Clause 5. The system of any of clauses 1-4, wherein the signal comprises a transcription factor that increases expression of the gene when the signal is above the threshold level.

Clause 6. The system of any of clauses 1-5, wherein the signal comprises a transcription factor that represses expression of the gene when the signal is above the threshold level.

Clause 7. The system of any of clauses 1-6, further comprising a vector containing the target site, a gene encoding the enzyme, and the gene encoding the HDR template.

Clause 8. A method comprising: exposing a cell to a timing indicator at a time, wherein the cell is configured to integrate a first homology directed repair (HDR) template into a double-stranded polynucleotide in response to exposing the cell to the timing indicator and to integrate a second HDR template into a repair formed by the first HDR template in the double-stranded polynucleotide in response to a molecular event; obtaining sequence data from the double-stranded polynucleotide after exposing the cell to the timing indicator and after the molecular event; and correlating the molecular event with the time based at least partly on analyzing the sequence data.

Clause 9. The method of clause 8, wherein the timing indicator comprises a change in light, a change in heat, a change in pH, availability of the first HDR template, or availability of an enzyme that creates a double strand break (DSB) in the double-stranded polynucleotide at a position configured for repair by the first HDR template.

Clause 10. The method of clause 8 or 9, wherein the molecular event is different than the timing indicator.

Clause 11. The method of any of clauses 8-10, further comprising exposing the cell to the timing indicator at a plurality of known times.

Clause 12. The method of any of clauses 8-11, wherein correlating the molecular event with the time includes identifying in the sequence data a sequence from the second HDR template adjacent to a first sequence from the first HDR template and adjacent to a second sequence from the first HDR template.

Clause 13. The method of any of clauses 8-12, wherein, in response to exposing the cell to the timing indicator, the cell is configured to upregulate expression of a gene encoding: the first HDR template, or at least a portion of an enzyme that creates a double strand break (DSB) in the double-stranded polynucleotide at a cut site that is configured to be repaired by the first HDR template.

Clause 14. The method of any of clauses 8-13, wherein the cell is further configured to iteratively integrate multiple copies of the second HDR template into the double-stranded polynucleotide while the molecular event continues.

Clause 15. A cellular system comprising: a molecule that degrades in the cellular system at a rate; a double-stranded polynucleotide; a first homology directed repair (HDR) template that is inserted into the double-stranded polynucleotide when the molecule is present in the cellular system at more than a threshold level; and a second HDR template that is inserted into the double-stranded polynucleotide when a signal caused by a molecular event is present.

Clause 16. The cellular system of clause 15, wherein the molecule comprises at least a portion of an enzyme configured to create a double strand break (DSB) in the double-stranded polynucleotide at a cut site flanked by regions homologous to the first HDR template.

Clause 17. The cellular system of clause 15 or 16, wherein the molecule comprises the first HDR template.

Clause 18. The cellular system of any of clauses 15-17, wherein the molecule comprises a transcription factor which increases transcription of a gene encoding the first HDR template or a gene encoding an enzyme configured to create a double strand break (DSB) in the double-stranded polynucleotide at a cut site flanked by regions homologous to the first HDR template.

Clause 19. The cellular system of any of clauses 15-18, wherein the molecule is ribonucleic acid (RNA) and the rate is determined in part by a 3′-poly(A) tail of the RNA.

Clause 20. The cellular system of any of clauses 15-19, wherein the second HDR template is inserted into a cut site introduced in the double-stranded polynucleotide by insertion of the first HDR template.

CONCLUSION

The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The term “based on” is to be construed to cover both exclusive and nonexclusive relationships. For example, “A is based on B” means that A is based at least in part on B and may be based wholly on B. By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

Certain embodiments are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. Skilled artisans will know how to employ such variations as appropriate, and the embodiments disclosed herein may be practiced otherwise than specifically described. Accordingly, all modifications and equivalents of the subject matter recited in the claims appended hereto are included within the scope of this disclosure. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, references have been made to publications, patents and/or patent applications (collectively “references”) throughout this specification. Each of the cited references is individually incorporated herein by reference for their particular cited teachings as well as for all that they disclose.