DNA backbone editing for DNA data storage

Methods of writing data to a DNA strand by inserting data-encoding oligos or symbols into a DNA backbone. One particular method of synthesizing a DNA strand encoding data includes cleaving a DNA backbone into multiple segments, and pasting a plurality of data-encoding oligo symbols between the multiple segments, with the terminal ends of the segments joining homologous terminal ends of the symbols, resulting in the DNA strand encoding data comprising alternating segments and symbols.

BACKGROUND

DNA is an emerging technology for data storage. DNA is used as a storage medium by converting binary code (0 and 1) to genetic code (A, C, G, T). The converted code can be synthesized into DNA molecules, which are then stored until read.

Current methods assert that a DNA strand or gene, to store 5 KB of data, can be written in 14 days. Comparatively, magnetic disk drives and magnetic tapes both can write 1 TByte in about an hour. A single DNA base pair location can store 2 bits; thus, 4000 Giga-base pairs would need to be stored in an hour to match the capabilities of a single disk drive or tape. Although current technology is believed to be capable of writing 15 base pairs an hour, there needs to be an 8 to 9 order of magnitude improvement in order for DNA data storage to be viable.

SUMMARY

This disclosure is directed to methods of writing data to a DNA strand by inserting data-encoding oligos into a DNA backbone.

One particular implementation described herein is a method of synthesizing a DNA strand encoding data. The method includes cleaving a DNA backbone into multiple segments (e.g., at least three segments), each of the multiple segments having terminal ends, and pasting a plurality of data-encoding symbols having terminal ends between the multiple segments, with the terminal ends of the segments joining homologous terminal ends of the symbols, resulting in the DNA strand encoding data comprising alternating segments and symbols. The method may further include cleaving the DNA strand encoding data comprising alternating segments and symbols into multiple second segments, each of the multiple second segments having terminal ends, and pasting a second plurality of data-encoding symbols having terminal ends between the multiple second segments, with the terminating ends of the second segments joining homologous terminal ends of the second symbols, resulting in a second DNA strand encoding data.

Another particular implementation described herein is a method that includes cleaving a DNA backbone into multiple segments by moving, via voltage, at least one of the DNA backbone and a Cas9 across a hydrophobic fluidic platform and combining on the platform the DNA backbone and the Cas9 resulting in the multiple segments each having terminal ends; and moving across the platform, via voltage, a plurality of data-encoding symbols having terminal ends on the platform and inserting the symbols between the multiple segments, with the terminal ends of the segments joining homologous terminal ends of the symbols, forming the DNA strand encoding data comprising alternating segments and symbols.

This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. These and various other features and advantages will be apparent from a reading of the following detailed description.

DETAILED DESCRIPTION

The amount of data generated by the global population is far greater than the amount of data storage space available. DNA is an incredibly compact data storage medium and is stable for many years in mild storage conditions. By converting binary code (0 and 1) to genetic code (A, C, G, T; as an example, A=00, C=10, G=01, and T=11), a DNA strand can be a biological storage media to hold the code.

As indicated above, various methods described herein edit a DNA backbone by inserting predetermined DNA symbols at intervals along the length of the backbone. A DNA symbol is an oligo, a short sequence of DNA (e.g., 1-20 bases) that encodes a unique segment of binary code. Alternately, a string of multiple nucleotides or a string of multiple symbols can be inserted. To insert a DNA nucleotide, symbol, or string of symbols into a DNA backbone, the backbone is cut in the desired insertion location and the nucleotide(s), symbol, or string of symbols is then pasted into the cut backbone. The methods allow the DNA code to be written in a massively parallel manner.

In the following description, reference is made to the accompanying drawing that forms a part hereof and in which is shown by way of illustration at least one specific implementation. The following description provides additional specific implementations. It is to be understood that other implementations are contemplated and may be made without departing from the scope or spirit of the present disclosure. The following detailed description, therefore, is not to be taken in a limiting sense. While the present disclosure is not so limited, an appreciation of various aspects of the disclosure will be gained through a discussion of the examples, including the figures, provided below. In some instances, a reference numeral may have an associated sub-label consisting of a lower-case letter to denote one of multiple similar components. When reference is made to a reference numeral without specification of a sub-label, the reference is intended to refer to all such multiple similar components.

As indicated above, for a data storage gene, each nucleotide is assigned a bit pattern; in one example, A=00, C=10, G=01, and T=11. In general, when an oligo is used in a data storage gene, the term “symbol” is used to represent the oligo that has a bit pattern. It is this “symbol” that is inserted into the DNA backbone. A symbol consists of a DNA sequence containing two linking ends with a base symbol between them. The base symbol is a DNA sequence (e.g., 1-20 pairs of nucleotides) that can be translated to a short sequence of binary code. There may be hundreds to thousands of different base symbols, depending on the length of the symbol.

In some implementations, other or additional information may be carried by the symbol. For example, the symbol may carry metadata, such as a timing indicator.

InFIG.1A, a schematic double strand DNA10is shown, calling out a first strand100acomprising nucleotides located between the3′ end and the5′ end and a complementary second strand100bcomprising complementary nucleotides located between the5′ end and the3′ end. The second strand100bis shown having a PAM sequence, which is a marker indicating a specific nucleotide sequence immediately following the target sequence. The PAM sequence is not part of the target sequence but is just downstream of the target sequence and indicates the relative location of the Cas9 activity. Even with the appropriate gRNA, if the PAM is not found, the Cas9 will not cleave the DNA. Similarly, if a PAM is found but a match for the gRNA is not found, the Cas9 will not cleave the DNA.

The DNA strand10shown is merely an example and does not represent any organism, as it is well known that DNA strands are significantly longer and that any nucleotide sequence may be present. The DNA backbone may be portions of, or the entirety of, e.g., a prokaryotic genome, a eukaryotic genome, a synthetic genome, or any combination thereof.

As described above, the present disclosure provides for editing of a DNA strand for the purpose of data storage by inserting DNA symbols at intervals along the length of the DNA backbone (e.g., into the DNA strand10). Specific biomolecules can cut either one or both strands of the DNA strand10at certain sequences. One of the more well-known DNA cleavage biomolecules is the Cas9 protein. The Cas9 protein is used together with a piece of RNA called a guide RNA (gRNA) that contains the desired target sequence to be cut. Other known DNA cleavage mechanisms are CRISPR/Cas (Clustered, Regularly Interspaced, Short Palindromic Repeats and CRISPR-associated) systems,Pyrococcus furiosusArgonaute (Pfago), TALEN (Transcription Activator-Like Effector Nuclease), restriction enzymes, zinc finger nucleases (ZFNs), or any biomolecule that can cleave DNA at a specific sequence.

When the Cas9 protein is mixed with the gRNA, the gRNA threads itself into the Cas9 protein. Because the gRNA contains the complementary sequence to the desired target sequence to be cut, the resulting Cas9-gRNA mixture searches the backbone of the DNA strand10for the PAM and a sequence matching its target gRNA. When it finds a match, the Cas9 cuts both strands of the DNA strand.

FIG.1Billustrates a process of cutting the DNA strand10by inserting a guide RNA (gRNA) strand threaded into a Cas9 protein (the combined molecule shown as102). When the gRNA strand binds to the DNA strand10, the attached Cas9 cuts the DNA strand10to form two strands100a,100b, the cleavage area indicated by the arrows. After cleaving the strands100a,100b, the gRNA strand and the complexed Cas9 protein102releases, leaving the cleaved strands100a,100b.

The DNA strand10and strands100a,100binFIG.1Aand the gRNA strand102ofFIG.1Bare shown in significantly simplified form to merely to provide the basis for the methods of this disclosure.

As indicated above, a data-encoding oligo or symbol is inserted into the cleaved DNA strand.FIG.2Aillustrates an example of an oligo or base symbol20that encodes data. Using the example above where A=00, C=10, G=01, and T=11, the base symbol20represents 11100100111110 in the top strand. The bottom strand is complementary to the top strand and generally does not encode any additional data; if any data were encoded in the bottom strand, it would generally be duplicative of the top strand. The base symbol20may be inserted into different locations on the backbone by attaching different linking ends to the base symbol20.

A cut DNA strand, such as that shown inFIG.1B, can be repaired by pasting in a DNA symbol that represents data, e.g., the base symbol20.

To insert or paste the data-encoding symbol20into the cut DNA strand, the symbol20must have sequences at both ends that are complementary to the cut ends of the DNA backbone. The symbol20may be a full double strand, as shown inFIG.2A, with blunt ends. In other implementations, the symbol may have chewed-back or overhanging ends that are complementary to overhanging ends on the cut DNA strand. Overhanging ends can be generated by digestion with an exonuclease or an endonuclease.

Different overhanging linking ends are shown added to the base symbol20inFIG.2Bresulting in the exemplary symbols200a,200b,200c,200d,200e,200f. Each of these symbols200has the same base symbol (the base symbol20) with different linking ends. In the particular example shown, each overhanging end is only two nucleotides; other examples of hanging ends can be shorter or longer, in most implementations however, longer. The sequences on the ends of the symbols are complementary to defined regions of the cut backbone.

Ends that are complementary can be joined together, e.g., via a homology-directed repair (HDR) process; in such a manner, a symbol200can be inserted into the cleaved DNA strand or backbone. Any HDR can be done via, e.g., Gibson assembly, restriction digest and ligation, ligase-independent cloning, sequence- and ligase-independent cloning, a “Golden Gate” method, Zero-Background Redα method, Hot Fusion method, BioBrick assembly, Seamless Ligation Cloning Extract method, overlap extension polymerase chain reaction, or other DNA assembly or cloning method. The resulting repaired DNA backbone now contains the desired inserted symbol(s).

The data-encoding symbols20and the symbols200having the linking ends, plus many others, can form a symbol library. Libraries of the base symbols20with every possible combination of linking ends (e.g., some of which are shown as the symbol200) may be created and stored until assembly.

Symbols with different linking ends, such as the symbols200, may be generated and stored in two different ways. In one implementation, base symbols with each possible combination of linking ends are synthesized and stored in a library for later use. In another implementation, symbols with different linking ends are created by attaching the desired terminal linking ends to the desired base symbols on-demand. With either method, the base symbols are inserted or pasted into a cleaved DNA strand.

FIGS.3A,3B and3Cschematically and stepwise show one implementation of a process for inserting encoded DNA symbols into a DNA strand or backbone to form a data storage strand by using already prepared symbols composed of base symbols with linking ends present.

InFIG.3A, a DNA backbone300is cleaved at four specific, unique and different sequence addresses, each proximate a PAM (depicted as vertical stripes), resulting in five backbone segments302. The result is a first segment302a, a second segment302b, a third segment302c, a fourth segment302dand a fifth segment302e. Although in this example the DNA backbone300is cleaved into five segments302, in other implementations the backbone300may be cleaved into, e.g., 3 to 100 segments, or even more than 100 segments. Typically, the backbone300is cleaved into at least 3 segments, or at least 5 segments, or at least 10 segments, or at least 20 segments, this cleaving being from the same process and being essentially simultaneously rather than sequentially.

The specific sequence addresses where the cleaving occurred are now split between adjacent segments300. For example, the sequence that identified the first cleavage location (depicted as diagonal hatching) resulting in the first segment302aand the second segment302bis now split between the first segment302aand the second segment302b; the sequence that identified the second cleavage location (depicted as horizontal lines) resulting in the second segment302band the third segment302cis now split between the second segment302band the third segment302c, etc.

InFIG.3B, the DNA symbols encoding data and containing ends homologous to the cleaved DNA backbone are introduced to the segments302. The five segments302a,302b,302c,302d,302eare shown inFIG.3Bwith a data-encoding symbol304positioned proximate each cleavage location; specifically, a first symbol304ais positioned to be bound to the first segment302aand the second segment302b, a second symbol304bis positioned to be bound to the second segment302band the third segment302c, a third symbol304cis positioned to be bound to the third segment302cand the fourth segment302d, and a fourth symbol304dis positioned to be bound to the fourth segment302dand the fifth segment302e. Each of the symbols304has a data-encoding base symbol305and ends that are complementary to the cleaved backbone so as to link the symbol304to the segments302; specifically, the first symbol304ahas a first base symbol305a, the second symbol304bhas a second base symbol305b, the third symbol304chas a third base symbol305cand the fourth symbol304dhas a fourth base symbol305d. The base symbol305may be the same or different from one symbol304to the other, however, the base symbol305is selected to provide the desired data bits. For example, there may be hundreds to thousands of different symbols304and different base symbols305. The number of base symbols305may be the same or different as the number of symbols304; as an example,FIG.2Ashows one base symbol20used to form six different symbols200inFIG.2B, by changing the terminal ends.

In some implementations, more than one symbol304may be positioned between two segments302. For example, two different symbols304, with homologous terminal ends, may be inserted adjoining each other between two segments302. If needed, an additional nucleotide or oligo may be included to facilitate the insertion of the multiple symbols304.

It is noted that in this example there are four symbols304(and four base symbols305) and five segments302, so that the number of symbols304is one less than the number of segments302. In other implementations, the number of symbols304can be greater than the number of segments302. In such an implementation, more than one symbol304can be inserted in one location between adjacent segments302. In another such implementation, one or more symbols304may be added to an end of the backbone, rather than at a cleaved location.

Homologous repair is shown inFIG.3Cto stitch the data-carrying symbols304and cleaved backbone segments302together. The resulting structure is a data storage strand310having the data-encoding base symbols305each located proximate a PAM in the DNA backbone.

It is noted that multiple processes may be done in series or in parallel (e.g., cleaving multiple backbones at a time, etc.) to increase the rate of synthesis of a large file of data.

FIGS.4A,4B,4C and4Dschematically and stepwise show another implementation of a process for inserting encoded DNA symbols into a DNA strand or backbone to form a data storage strand. In this process, the symbols with different linking ends are created by attaching of desired terminal linking ends to the desired base symbols on-demand. A base symbol encoding the desired data bits is selected from a library, and the desired linking ends are selected from a second library. The linking ends and the base symbols contain a small homologous region to enable their joining via HDR. Assembling base symbols and linking ends on-demand reduces the required library size and simplifies liquid handling.

InFIG.4A, as in the previous process, a DNA backbone400is cleaved at four, specific, unique and different sequence addresses, each proximate a PAM (depicted as vertical stripes), resulting in five backbone segments402. The result is a first segment402a, a second segment402b, a third segment402c, a fourth segment402dand a fifth segment402e. The specific sequence addresses, where the cleaving occurred, are now split between adjacent segments402. For example, the sequence that identified the first cleavage location (depicted as diagonal hatching) resulting in the first segment402aand the second segment402bis now split between the first segment402aand the second segment402b.

InFIG.4B, four base symbols405a,405b,405c,405dencoding the desired data are shown; the base symbols405may be the same or different but are selected to provide the desired data bits. Also shown are linking terminal ends403for attachment to the base symbols405; each base symbol405has two linking ends403, one for each end. Specifically, the first base symbol405ahas first linking ends403a, the second base symbol405bhas second linking ends403b, the third base symbol405chas third linking ends403c, and the fourth base symbol405dhas fourth linking ends403d. These linking ends403may be held in a library formed of numerous oligos and selected based on the nucleotides needed to provide the engagement between the base symbols405and the segments402.

When combined, the base symbols405and the linking ends403form a symbol404(called out inFIG.4C); thus, each of the symbols404has one data-encoding base symbol405and ends403that are complementary to the cleaved backbone segments so as to link the symbol404to the segments402. Specifically, the first base symbol405alinks to the first ends403a, the second base symbol405blinks to the second ends403b, the third base symbol405clinks to the third ends403c, and the fourth base symbol405dlinks to the fourth ends403d.

InFIG.4C, the resulting symbols404, composed of the DNA base symbols encoding data linked to the linking ends, are introduced to the backbone segments. The five segments402a,402b,402c,402d,402eare shown with a data-encoding symbol404positioned proximate each cleavage location; specifically, a first symbol404ais positioned to be bound to the first segment402aand the second segment402b, a second symbol404bis positioned to be bound to the second segment402band the third segment402c, a third symbol404cis positioned to be bound to the third segment402cand the fourth segment402d, and a fourth symbol404dis positioned to be bound to the fourth segment402dand the fifth segment402e. Each of the symbols404has the base symbol405and the linking ends403.

The resulting structure is a data storage strand410inFIG.4Dhaving the data-encoding base symbols405located proximate a PAM in the DNA backbone.

The data storage strands310,410can be read by any suitable mechanism to identify (sequence) the nucleotides and decipher the data.

Alternately, these data storage strands310,410can undergo an additional cut-and-paste operation, such as shown inFIGS.3A-3Dand/orFIGS.4A-4Dto insert additional data-encoding symbols. These subsequent insertion points may be within a strand of the original backbone300,400or may be within the symbols304,404that were previously added to the original backbone300,400. The same or different DNA strand cleaving methodology (e.g., Cas9) may be used for any subsequent or additional operation.

By knowing the relative location of the data storage base symbols305,405in relation to a marker, such as the PAM, the data can be read from the DNA strand by any suitable means. Alternately, any of the backbone segment, the backbone segment plus linking ends, or any linking ends can be markers identifying the location of the data. In addition to including a marker, the DNA backbone can carry additional information such as time stamps, indexes, or other formatting to aid in computational based tasks.

The previous discussion has provided an example utilizing a library of data-encoding symbols and a library of linking ends to form a DNA strand having the data-encoding symbols positioned in the strand in an arranged desired order. By utilizing multiple symbols and multiple linking ends, all of which are predetermined oligos, and utilizing parallel reactions, the synthesis rate of the final DNA data strand can be greatly improved compared to a de novo gene synthesis where each nucleotide is added one at a time.

In such a manner, content (e.g., a document, a picture) can be saved as data on DNA. The content can be written on one or more DNA strands or backbones; if multiple strands or backbones are used, they do not need to be similar in length or in content. DNA strands or backbones with different data written to them, that are part of the same file, can be mixed together or stored separately. This process can be repeated, e.g., by cutting the symbols that have been inserted, and inserting into the cut location a second (e.g., different) symbol.

The backbone cleaving synthesis methods described above can be implemented in any manner, e.g., utilizing various reactors, flasks, beakers, etc. The method is also particularly suited to be done as a microfluidic lab-on-a-chip process.

Lab-on-a-chip is a common term for an integrated circuit (“chip”) on which one or several laboratory functions or chemical reactions are done. The chip can be no more than a few square centimeters. Labs-on-a-chip handle extremely small fluid volumes (e.g., measured as pico-liters) and are often called microfluidic systems. In digital microfluidics, the lab-on-a-chip has a hydrophobic “chip platform” on which fluid droplets (e.g., liquid droplets) can be manipulated by precisely controlled voltage application. The platform may have a cover plate covering the fluidic area. By utilizing the feature of surface tension of the fluid on the platform, the fluid can be precisely moved across the platform by voltage applied to the platform, e.g., in a grid.

For the synthesis methods described above, the lab-on-a-chip is operably and fluidically connected to the ingredients or components (e.g., the DNA backbone, the Cas9 protein and the gRNA, or the Cas9-gRNA mixture, and the symbols for the first method described above in relation toFIGS.3A,3B and3C, or the DNA backbone, the Cas9 protein and the gRNA, or the Cas9-gRNA mixture, the base symbols, and the linking ends for the second method described above in relation toFIGS.4A,4B,4C and4D). Each of the components is retained in a well or other liquid storage compartment. In some designs, there may be only one well for the DNA backbone, one well for the Cas9-gRNA mixture, at least 10,000 wells for the symbols, or at least 20,000, or at least 30,000 wells, or at least 65,000 wells. Additionally or alternately, there can be at least 120 wells for the linking ends, or at least 30 wells, or at least 60 wells.

Using known techniques (e.g., voltage differential on the platform), the dispensed components (e.g., DNA backbone, base symbols, etc.) are moved on (across) the platform and mixed in the desired steps. All mixing of the components can be done on the platform or a dedicated mixing station may be used for one or more of the steps, e.g., utilizing heat and/or agitation, e.g., for cleaving the DNA backbone with the Cas9 and gRNA. In some implementations, the platform may include a controllable reaction facilitator, such as a UV light source, and/or the final mixing station may include a voltage source, e.g., to align the completed data encoded strand to aid in collection.

One suitable (physical) size for a lab-on-a-chip is about 20 mm by 20 mm, which is compatible to an 8 inch wafer and could have 785,000 array elements, each array element having controllable voltage independently applied thereto. In some implementations, each well or other storage compartment is 10× the size of an array element. This would provide 66,560 wells and leave 119,000 arrays for transport and mixing of the components on the platform.

A stacked or otherwise three-dimensional array of labs-on-a-chip would increase density and decrease required area for the synthesis. A droplet elevator could be used to provide synthesis on multiple vertically stacked levels.

A cleaning or decontamination mechanism may be included in the lab-on-the-chip to rinse, wash, or otherwise decontaminate certain or all grid locations that have had or will have a component present thereon. For example, an amount (e.g., droplet) of cleaning solution (e.g., hydrogen peroxide) can be applied to and moved across the platform to cleanse the platform. In one particular example, the cleaning solution can follow immediately behind, e.g., a base symbol, thus cleaning and decontaminating the surface of any oligo that may remain. In another particular example, the cleaning solution can trace the path the oligo will follow.

FIGS.5A and5Billustrate two steps of an example synthesis method.

FIGS.5A and5Bshow a lab-on-a-chip500with a platform working surface502having numerous cells each configured for independently receiving a voltage. The lab500includes a plurality of wells504for the components. Although the figures show the wells504on two opposite sides of the platform502, the wells504may be arranged on the chip500in any location and in any order. The lab500also has a final mixing location506for the final mixing or synthesis step (e.g., shown inFIG.3CorFIG.4D) for the data storage gene.

In a first step, shown inFIG.5A, a Cas9 protein from the well512is combined at a first location on the platform502with a gRNA molecule from the well514. The selected components (e.g., Cas9 protein, gRNA) are moved via voltage on the platform502to meet and combine. In a second step, also shown inFIG.5A, the combined Cas9-gRNA is moved to a second location where it is mixed with DNA backbone from the well510. At this second location, the Cas9-gRNA cleaves the DNA backbone.

InFIG.5B, the cleaved backbone segments are moved to the final mixing location506where, in this example, four data-encoding symbols from four wells516a,516b,516c,516dare mixed with the backbone segments. After mixing, the result is a reunited DNA strand with the data-encoding symbol inserted into the strand.

The lab500also includes a PCR region520to replenish the linker and/or symbol libraries, the PCR region520including wells for PCR chemicals530a,530band a PCR station540. As the data-encoding symbols are depleted with each synthesized strand, occasionally, the symbols need to be replenished; the PCR region520of the lab500allows this replenishment to be done at the lab500.

Because of the need to move numerous components, including the symbols, across the platform502, to the final mixing location506, and to the PCR region520, many of which are moved or moving simultaneously, numerous paths are used. For example, at a point in time, one hundred symbols, at least one of which is a unique symbol, may be moving on the platform502as well as the segment backbone. In most implementations, these paths are not constrained by channels or other physical or set paths on the platform502, but movement of the fluids on the platform502is controlled merely by the applied voltage. It is noted that due to the large number of paths needed, a very detailed and complicated traffic map may be needed.

The above specification and examples provide a complete description of the structure and use of exemplary implementations of the invention. The above description provides specific implementations. It is to be understood that other implementations are contemplated and may be made without departing from the scope or spirit of the present disclosure. The above detailed description, therefore, is not to be taken in a limiting sense. While the present disclosure is not so limited, an appreciation of various aspects of the disclosure will be gained through a discussion of the examples provided.

Unless otherwise indicated, all numbers expressing feature sizes, amounts, and physical properties are to be understood as being modified by the term “about,” whether or not the term “about” is immediately present. Accordingly, unless indicated to the contrary, the numerical parameters set forth are approximations that can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings disclosed herein.

Spatially related terms, including but not limited to, “bottom,” “lower”, “top”, “upper”, “beneath”, “below”, “above”, “on top”, “on,” etc., if used herein, are utilized for ease of description to describe spatial relationships of an element(s) to another. Such spatially related terms encompass different orientations of the device in addition to the particular orientations depicted in the figures and described herein. For example, if a structure depicted in the figures is turned over or flipped over, portions previously described as below or beneath other elements would then be above or over those other elements.

Since many implementations of the invention can be made without departing from the spirit and scope of the invention, the invention resides in the claims hereinafter appended. Furthermore, structural features of the different implementations may be combined in yet another implementation without departing from the recited claims.