CRISPR-Cas12a-based plasmid pool DNA database management method and system

Abstract

The invention discloses a CRISPR-Cas12a-based plasmid pool DNA database management method and a CRISPR-Cas12a-based plasmid pool DNA database management system. The method comprises the following steps: S1, data writing: encoding data required to be stored by a user, assembling an encoded DNA sequence and a designed multi-layer address sequence into a plasmid map, and synthesizing and cloning to form a plasmid pool database; s2, data retrieval: S21, analyzing a target address; s22, assembling the one-way guide RNA and a Cas12a protein into a ribonucleoprotein complex; s23, carrying out specific cleavage on the plasmid pool database by utilizing a ribonucleoprotein complex; s24, enriching a cutting product; s25, performing amplification and sequencing on the enriched product, and restoring target data after decoding; s3, data deletion: S31, designing a one-way guide RNA (Ribonucleic Acid) aiming at a to-be-deleted data address sequence; s32, cutting a target plasmid through a Cas12a protein; and S33, separating and removing the cut plasmids through a gel electrophoresis method so as to realize data deletion, and promoting DNA storage from a static archiving mode to a dynamic database management mode.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and information storage technology, and in particular to a management method and system for encoding, storing, retrieving, adding, and deleting DNA databases based on a plasmid pool architecture using the CRISPR-Cas12a system. Background Technology

[0002] With the explosive growth of global data volume, traditional silicon-based storage technologies face severe challenges in terms of capacity, energy consumption, and long-term preservation. Deoxyribonucleic acid (DNA), due to its extremely high theoretical storage density (reaching the EB / mm³ level) and stability lasting up to a thousand years, is considered the most promising next-generation massive data storage medium. Currently, DNA data storage technology has achieved full-process validation from data encoding and synthesis to sequencing and decoding.

[0003] However, most existing DNA storage systems are based on a "one-pot" mixed DNA oligo pool, which functions more like a static archive tape. They lack the ability for dynamic, fine-grained data management, namely efficient random access, targeted deletion, and update capabilities. This has become a core bottleneck hindering their practical application. Existing technologies for achieving random access mainly fall into two categories: one is amplification retrieval based on polymerase chain reaction (PCR), which is limited by primer specificity and amplification bias, and cannot achieve physical deletion of data; the other is retrieval based on physical partitions (such as microcapsules or silica spheres), which uses barcodes or spatial addressing, but sacrifices DNA storage density, and its capacity is limited by the number of partitions.

[0004] Recent studies have explored manipulating DNA data using the CRISPR-Cas system, such as rewriting information within cells or using Cas12a for keyword recognition. However, these methods have failed to achieve efficient, precise, and programmable full-scale operations (including reading, deleting, and copying) on ​​specific data molecules within massive DNA data pools. Furthermore, plasmids, as stable and high-capacity DNA vectors, have not yet been systematically integrated into a scalable physical architecture that supports full database operations.

[0005] Therefore, there is an urgent need in this field for a new paradigm of DNA data storage system that can support efficient random access, dynamic data management, and has extremely high theoretical scalability. Summary of the Invention

[0006] This invention aims to address the shortcomings of existing technologies. The technical problem it seeks to solve is how to provide a DNA database management solution capable of high-density storage, high-speed random retrieval, and precise data deletion and copying of massive amounts of DNA data. This overcomes the limitations of existing "one-size-fits-all" DNA storage methods, which lack dynamic management capabilities, and the low density of physical partitioning storage methods. Specifically, it addresses how to combine the specific cutting capabilities of CRISPR-Cas12a with the characteristics of plasmid vectors to construct a physical system that supports "create, delete, modify, and search" operations similar to an electronic database.

[0007] The technical problem of this invention is solved by the following technical solution: In a first aspect, the present invention provides a method for managing a CRISPR-Cas12a-based plasmid-of-pool (POP) DNA database, comprising the following steps: S1. Data writing steps: Encode the data that the user needs to store, assemble the encoded DNA sequence with the designed multilayer address sequence into a plasmid map, and form a plasmid pool database after synthesis and cloning. S2, Data retrieval steps, specifically including: S21. Based on the user's query request, parse out the target address; S22. Design a single-guide RNA corresponding to the target address, and assemble the single-guide RNA with Cas12a protein into a ribonucleoprotein complex. S23. The ribonucleoprotein complex is used to specifically cut the plasmid pool database under suitable reaction conditions to label the target plasmid and generate sticky ends at the address sequence of the target plasmid. S24. Enrich the cleavage product based on the marker (i.e., the target plasmid); S25. Amplify and sequence the enriched products, and then decode to restore the target data; S3, Data Deletion Steps, specifically include: S31. Design a single-guide RNA for the address sequence of the data to be deleted; S32. The target plasmid is cleaved by the Cas12a protein; S33. Data deletion is achieved by separating and removing the cleaved plasmids using gel electrophoresis.

[0008] In some embodiments, the following technical features are also included: In step S1, the data types that need to be stored include binary data information and biological genetic information.

[0009] In some embodiments, the encoding method in step S1 includes, but is not limited to, modulation coding, Huffman coding, Base64 coding, or fountain coding.

[0010] In some embodiments, in step S1, the plasmid pool database consists of multiple plasmids, each plasmid having a uniform length; each plasmid contains at least one address sequence for addressing.

[0011] In some embodiments, the plasmid includes a fluorescent recognition element or an anti-resistance protein expression element.

[0012] In some embodiments, in step S1, the construction of the plasmid pool database may be based on a hierarchical model, a network model, or a relational model.

[0013] In some embodiments, in step S23, the marking is achieved by cutting to generate sticky ends and connecting them with a connector complementary to the sticky ends; the suitable reaction conditions include: the reaction buffer containing a reaction solution suitable for the Cas12a protein and a reagent for inhibiting RNA degradation.

[0014] In some embodiments, the Cas12a protein includes, but is not limited to, Cas12a proteins and variants from different sources such as AmCas12a, FnCas12a, and LbCas12a.

[0015] In some embodiments, in step S2, the data manipulation forms in the data retrieval step include single address access, single file access, batch data access, bidirectional access, and logical access.

[0016] Secondly, the present invention provides a DNA database management system for implementing the above-described method, the system comprising: The logic management layer is configured to receive user data manipulation instructions and parse them into unidirectional guide RNA design instructions for specific address sequences. The physical operation layer includes: The plasmid pool database consists of multiple plasmids containing encoded data and multi-layer address sequences; The CRISPR-Cas12a Oligonucleotide Retrieval Engine (CORE) is configured to perform specific cleavage and enrichment operations based on single guide RNA and Cas12a protein.

[0017] In some embodiments, the logical management layer is configured to use an entity-relationship model to abstract and manage the plasmid pool database, and may also be compatible with other database structures such as hierarchical databases.

[0018] In some embodiments, the CRISPR-Cas12a retrieval engine includes: The targeted recognition and cleavage module, composed of a ribonucleoprotein complex consisting of a programmable single guide RNA and Cas12a protein, is used to specifically recognize and cleave the address sequence on the target plasmid to generate sticky ends. The specific enrichment module is configured to achieve specific capture and enrichment of the target plasmid based on the cutting operation; In some embodiments, the specific enrichment module consists of a DNA ligase and a adapter sequence that is completely complementary to the sticky end, for the purpose of specific capture and enrichment of the target fragment.

[0019] The beneficial effects of this invention compared to the prior art include: This invention innovatively applies the specific gene-editing capabilities of the CRISPR-Cas12a system to the retrieval and management of DNA data storage. Utilizing its programmable single-path guide RNA, it precisely targets and cuts specific address sequences. Combined with the physical properties of conformational changes in plasmid vectors after cutting, gel electrophoresis is used to separate cut and uncut plasmids. This synergistic approach achieves an integrated system of "directional addressing - precise manipulation - physical separation" for specific data within a massive DNA data pool. This technological combination fundamentally solves the core challenge of existing DNA storage technologies in achieving efficient random access and physical deletion, advancing DNA storage from a static archiving model to a dynamic database management model.

[0020] Specifically, the specific cleavage of the address sequence by the Cas12a protein-guideRNA complex ensures operational precision and avoids interference from non-target data; the difference in migration rates between the linearized plasmid and the closed circular plasmid generated by cleavage in gel electrophoresis provides the basis for physical separation, thereby achieving complete data deletion; and the combination of complementary adapter ligation and enrichment greatly improves the sensitivity and signal-to-noise ratio of target data retrieval. This organic combination of "biochemical recognition" and "physical separation" is the core principle behind this scheme's efficient dynamic management.

[0021] In addition, some embodiments also have the following beneficial effects: By using plasmids of uniform length as storage units and integrating multi-layer address sequences on each plasmid, a hierarchical plasmid pool architecture is constructed, which enables the database address space to expand exponentially. The theoretical storage capacity can be increased to the YB (Yottabyte, 1 YB = 10^24 Bytes) level, meeting the needs of future ultra-large-scale data storage.

[0022] By using an entity-relational model to abstract physical storage through a logical management layer, this system can be compatible with and understand multiple database models (hierarchical, network, relational), providing users with a user-friendly and powerful data operation interface, and improving the system's practicality and flexibility.

[0023] By setting up fluorescent recognition elements or resistance protein expression elements, it is easier to enrich and identify plasmids containing target data during in vivo preservation and screening, thus increasing the system's application scenarios and operability.

[0024] By selecting different Cas12a protein variants (such as AmCas12a, FnCas12a, and LbCas12a), different PAM (Protospacer Adjacent Motif; for Cas12a, its PAM sequence is typically 5'-TTTV-3' (where V is A, C, or G)) sequence requirements and reaction conditions can be adapted, improving the system's compatibility and customizability.

[0025] Other beneficial effects of the embodiments of the present invention will be further described below. Attached Figure Description

[0026] Figure 1 This is a flowchart of a plasmid pool DNA database management method based on CRISPR-Cas12a in an embodiment of the present invention; Figure 2 This is a schematic diagram of the overall architecture of the POP database based on CORE in an embodiment of the present invention; Figure 3 This is a database address structure design diagram in an embodiment of the present invention; Figure 4 This is a flowchart of database operations in an embodiment of the present invention; Figure 5 This is a schematic diagram of the "cut-and-connect" dual recognition mechanism of the CRISPR-Cas12a retrieval engine in an embodiment of the present invention; Figures 6a to 6g This is a screenshot of the plasmid information sequence in an embodiment of the present invention; Figure 7 This is a conceptual diagram of parallel database operations in an embodiment of the present invention.

[0027] Figure 8 This is a schematic diagram illustrating the improvement of database address capacity and overall capacity under the CORE mechanism in this embodiment of the invention. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and preferred embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other.

[0029] The core of this invention lies in constructing a physical storage architecture based on a plasmid pool and utilizing the CRISPR-Cas12a Oligonucleotide Retrieval Engine (CORE) to manage the data within this architecture. The POP architecture uses circular plasmid DNA as its core vector, with each plasmid serving as an independent storage unit containing the data sequence and an address sequence for addressing. The CORE engine uses a programmable single guide RNA (sgRNA) to guide the Cas12a protein to cleave specific address sequences, thereby enabling data retrieval and deletion operations through biochemical methods (such as ligation enrichment) or physical methods (such as electrophoretic separation).

[0030] To address the chaotic "one-size-fits-all" storage structure of existing DNA databases, this invention proposes a physical structure design architecture for DNA databases based on plasmid characteristics. To address the lack of efficient single-molecule search tools, this invention proposes a CRISPR-Cas12a-based Core Search Engine (CORE) retrieval mechanism. Based on this, this invention provides a plasmid pool (POP) DNA database management system architecture based on the CORE retrieval mechanism. The main objective of this invention is:

[0031] A high-density, high-stability DNA database physical storage architecture (POP framework) is provided. It provides a fast, accurate, and programmable single-molecule DNA retrieval engine (CORE) that supports random access to DNA databases and data addition and deletion. This paper provides a multi-level hierarchical addressing method that supports exponential expansion, breaking through the limitations of traditional addressing modes on storage capacity. It provides a complete DNA database solution encompassing physical structure, logical structure, retrieval methods, and operational mechanisms.

[0032] Example 1: This embodiment aims to solve the problem that "one-pot" DNA storage cannot perform targeted retrieval and physical deletion, and provides a CRISPR-Cas12a-based plasmid pool DNA database management method, such as... Figure 1 As shown, the specific steps include: S1. Data writing step. The text data to be stored, namely "plum", "orchid", "bamboo", and "chrysanthemum" (abbreviated as M, L, Z, J respectively), is first converted into a binary data stream. This binary stream is encoded using modulation coding: every 2-bit binary number is mapped to a base (A, T, C, G). Subsequently, primer sequences are randomly selected from a four-base modulation primer library with a 50% GC content, and an exclusive OR operation is performed with the original coding sequence to adjust the global GC content, and secondary modulation is performed on the possibly formed consecutive identical bases. The encoded data sequence is assembled with a pre-designed two-layer address sequence into 4 complete plasmid maps. The size of each plasmid is designed to be uniformly 2760 bp. The plasmid maps are sent to a commercial synthesis company for synthesis, and then transformed into Escherichia coli for cloning, proliferation, and preservation, ultimately forming a plasmid pool database.

[0033] S2. Data retrieval step. When the user requests to retrieve the data of "plum":

[0034] S21. The logical management layer analyzes the secondary address sequence corresponding to the "M" data block.

[0035] S22. Design an sgRNA that is completely complementary to this address sequence. The chemically synthesized sgRNA is incubated with the AmCas12a protein in a buffer at room temperature for 10 minutes to form a ribonucleoprotein (RNP) complex.

[0036] S23. According to the recommended reaction conditions of the used Cas12a protein variant or the optimized time determined through preliminary experiments, usually incubate for 5 - 30 minutes to ensure complete cleavage. Take an appropriate amount of the solution containing the POP database, mix it with the prepared RNP complex, and incubate at 37 °C for 30 minutes. The AmCas12a protein will recognize and cleave the target address sequence near its PAM sequence (5'-TTTV-3'), generating sticky ends at the cleavage site.

[0037] S24. Add T4 DNA ligase and an adapter sequence that is completely complementary to the above-mentioned sticky ends (the 5' end of this adapter sequence is pre-phosphorylated and has a universal sequencing primer binding site) to the reaction system. Connect at 16 °C for 1 hour. This step realizes the specific capture and enrichment of the cleavage products through the base complementary pairing principle and the ligation characteristics of T4 enzyme.

[0038] S25. Using the ligation product as a template, perform PCR amplification with primers targeting the universal primer binding site. Perform next-generation sequencing (NGS) on the PCR product, and decode the measured base sequence to restore the original text data of "plum".

[0039] S3. Data deletion step. When the user requests to delete the data of "chrysanthemum":

[0040] S31: Design sgRNA targeting the address sequence of the “J” data block.

[0041] S32: The RNP complex composed of sgRNA and Cas12a protein was used to cut the POP database, causing all plasmids containing "J" data to change from a supercoiled closed-loop structure to a linear structure.

[0042] S33: The entire system after cutting was subjected to 1% agarose gel electrophoresis. Under the influence of an electric field, the linearized plasmids and the uncut closed circular plasmids separated due to their different migration rates. The linearized plasmid bands after cutting could be physically removed from the gel and discarded, thus achieving complete data deletion. The remaining closed circular plasmids could be recovered from the gel to obtain a new POP database with the target data deleted.

[0043] The working principle of this embodiment is as follows: The CORE engine enables precise targeting to specific addresses through the programmability of sgRNA; the cleavage activity of Cas12a linearizes the target plasmid, which not only provides a basis for specific enrichment based on sticky ends, but also creates conditions for its physical separation from closed circular plasmids; ligation enrichment and gel electrophoresis amplify and realize this distinction from biochemical and physical levels, respectively, and finally complete the retrieval and deletion operations.

[0044] Example 2: This embodiment provides a database management system that utilizes the CRISPR-Cas12a retrieval engine (CORE) to perform random access and targeted addition / deletion of DNA databases based on a plasmid pool (POP) architecture. Figure 2 As shown, the system workflow mainly includes two paths: Writing and Assembly Path: First, data is written (supporting binary or FASTA format) and read (supporting random access and editing functions such as adding, deleting, and copying). Then, the data enters the database management center, where it undergoes segmentation, encoding, address logic, and sequence assembly to generate a plasmid map. The map information is sent to the synthesis platform to complete the synthesis of plasmid pools containing specific physical structures (such as POP structures). The synthesized plasmid pools can be accessed by address using the CORE retrieval system, and finally, data is output through sequencing and decoding.

[0045] Target reading path: Users can also submit target sequences directly from the database management center, and the target data can be directly located and output through the CORE retrieval system.

[0046] Furthermore, there is a two-way linkage between plasmid pool synthesis and database storage (using dry storage or partitioned storage strategies), enabling synchronous updates of the material library and information database. Throughout the entire process, core steps such as plasmid pool synthesis, CORE retrieval, database storage, and data output can be repeatedly performed through an automated work platform.

[0047] The management methods employed by a database management system specifically include the following steps: Step 1: Data Writing. The data that the user needs to store is encoded, and the encoded DNA sequence is assembled with the designed multilayer address sequence to form a plasmid map. After synthesis and cloning, a POP database is formed.

[0048] Step 2: Data Storage. The synthesized POP database is processed through pooling, morphological manipulation, and drying to meet actual storage requirements and ensure long-term storage of DNA information.

[0049] Step 3: Data Retrieval. Based on the user's query request, the database management layer parses the target address; designs the corresponding sgRNA (single guide RNA), assembles it with Cas12a protein into a complex, and specifically cleaves the POP database under suitable temperature and solution conditions to generate sticky ends; enriches the data by ligation through complementary adapters, amplifies the enriched product by PCR, and sequences it. After decoding, the target data is restored.

[0050] Step 4: Data Deletion. Design an sgRNA targeting the address sequence of the data to be deleted, and cut the target plasmid with Cas12a. The uncuttered plasmid and the cut plasmid can be physically separated by gel electrophoresis, thereby realizing the deletion of data and the recovery of undeleted data.

[0051] Step 5: Data Copying. The data to be copied is replicated on a large scale by transforming plasmids into bacteria such as E. coli.

[0052] The data types to be stored include, but are not limited to, binary data information and biological genetic information. Any information that can be stored using DNA base sequences can be stored in the POP database. The encoding methods include, but are not limited to, modulation encoding, Huffman encoding, Base64, and fountain codes.

[0053] The POP database consists of plasmids, and the plasmid pool for each detection unit consists of plasmids of equal length; each plasmid has at least one address sequence to meet addressing requirements.

[0054] The plasmid backbone of the POP plasmid library can be designed in a variety of ways, including but not limited to adding fluorescent recognition elements, resistance protein expression elements, etc., so as to meet the diverse needs of data preservation and screening in vivo.

[0055] The POP database construction rules include, but are not limited to, hierarchical models, network models, and relational models.

[0056] The physical preservation method for POP data pools described herein is compatible with various in vivo and in vitro storage methods, including but not limited to silica encapsulation, hydrogel microsphere encapsulation, microcapsule sealing, DNA drying, and transfer into live bacterial cells.

[0057] The data retrieval tool CORE uses CRISPR proteins for data identification and cleavage. The proteins used include, but are not limited to, Cas12a, and the protein types include Cas12a variants from various sources.

[0058] The CORE-based data cutting method is based on the sgRNA protein complex cutting of specific address sequences, and the data separation method includes, but is not limited to, agarose gel electrophoresis for data separation and extraction.

[0059] The data retrieval process includes complete data addition and deletion as well as random access. Depending on the database design pattern, the data manipulation methods include, but are not limited to, query, insertion, deletion, update, single address access, single file access, batch data access, bidirectional access, and logical access.

[0060] The data copying method described above uses plasmid replication based on intracellular transformation, and the media used include, but are not limited to, Escherichia coli and yeast.

[0061] The data retrieval and deletion reaction conditions involve manipulating plasmid DNA stored in vitro. The reaction buffer may contain a reaction solution suitable for the corresponding Cas12a protein, a reagent to inhibit RNA degradation, and the corresponding Cas12a and sgRNA complex. The Cas12a protein types used include, but are not limited to, various protein variants such as AmCas12a, FnCas12a, and LbCas12a.

[0062] Example 3: This embodiment provides a plasmid pool database management system based on the CRISPR-Cas12a search engine, including: The Plasmid Pool Physical Storage Architecture (POP framework) uses circular plasmid DNA as the core data storage medium. Each plasmid can function as an independent "page," containing one or more encoded data "blocks." Each plasmid contains at least one address sequence for single-molecule retrieval. Each file or each file pool can be designed to carry plasmid pools of the same length of data, facilitating band separation based on pre- and post-cut size. The circular structure of a single plasmid provides natural physical isolation for each address. This physical storage architecture allows for in vitro solution or desiccation, as well as in vivo (e.g., bacteria, cells) storage with multiple copies or genome replication.

[0063] The CRISPR-Cas12a oligonucleotide search engine (CORE) is the core processing unit of the database. This unit consists of a targeted recognition and cleavage module and a specific enrichment module. The targeted recognition and cleavage module is an RNP (ribonucleoprotein) complex composed of programmable sgRNA and Cas12a protein (preferably AmCas12a and its variants, but Cas12a from other sources is also acceptable), used to specifically recognize and cleave the address sequence on the target plasmid. The most common cleavage site type, located near the PAM side, is between positions 20 and 23. Different sources of Cas12a protein, sgRNA structure, methylation, reaction solutions, etc., may have some influence on the distribution concentration of cleavage types, as long as the sticky end of the cleavage site on the PAM side protrudes by ≥2 bases. The specific enrichment module uses DNA ligase to ligate a tag sequence with a linker that is completely complementary to the above-mentioned sticky ends to the cleavage product, achieving specific capture and enrichment of the target fragment. Preferably, the ligation process requires the adapter sequence to have at least two bases that are complementary to the sticky end, more preferably three to five bases; adapters with three prominent bases have the highest ligation efficiency. The CORE engine can complete the process in 10 minutes at 37°C using the aforementioned dual recognition mechanism of "cut-ligation". The specifically enriched target fragment can be read using sequencing (Sanger, NGS, nanopore, etc.) to retrieve the loaded data.

[0064] Data encoding and a multi-layered logical addressing architecture are established. This database system is compatible with various DNA storage encoding methods, with the constraint being the length of information carried by a single address. Each data block has two segments containing address sequences with specific information. An example of the database address structure design is shown below. Figure 3 As shown, the locations of sgRNAs (including sgRNA MLZJ and sgRNAs IN and M), the localization points of sgRNAs (101, 102, 111, 112, 121, 122), and a simplified diagram of each sgRNA are marked. Their core structure and related components are as follows:

[0065] 1. Core Address and Data Area Position pointer: Figure 3 In the diagram, 11 represents Pointer 1 and 12 represents Pointer 2, which are used for precise location in the database.

[0066] Figure 3 21 indicates the PAM sequence: located near the pointer, it provides the necessary specific sequence context for the recognition and binding of subsequent CRISPR systems (such as the Cas12a and sgRNA complex).

[0067] Data storage area: Figure 3 31 represents Data, which is a DNA fragment approximately 200 nucleotides (nt) in length used to actually encode and store target information.

[0068] 2. sgRNA type identification Figure 3 In the diagram, 101 represents sgRNA M / L / Z / J1, and 102 represents sgRNA M / L / Z / J2. Their types are explained below:

[0069] M: "Plum" in Example 1 L: "Lan" in Example 1 Z: Bamboo in Example 1 J: In Example 1, "chrysanthemum" 3. Address sequence identifier To locate the address of this data information, an address sequence was designed: Address sequence: Figure 3 41 in the middle indicates P-IN: representing the first-level address sequence, or simply "address IN".

[0070] Figure 3 In the middle, 42 indicates PM: representing a second-level address sequence, abbreviated as "address M".

[0071] CORE's orthogonal partitioning mechanism allows for hierarchical address sequence design. By designing N layers of addresses, the system can achieve a theoretical addressing capacity of (420)N, realizing an exponential expansion of the address space. According to the existing mechanism, it supports address design of up to 16 layers, thereby enabling the theoretical capacity of a single pool to reach the 1000-byte level.

[0072] Database logical management layer. It uses the Entity-Relational (ER) model to abstract physical storage, defining logical entities such as "plasmids" and "data blocks" and their corresponding relational logic according to actual database requirements. The data management layer is responsible for receiving user data operation commands (create, delete, modify, query), parsing them into sgRNA designs for specific address sequences, and calling the CORE to execute the physical operations.

[0073] Figure 2 This demonstrates the complete workflow from the user data layer and logic management layer to the POP physical layer and CORE engine. The DNA database logical operation method includes the following steps:

[0074] Step 1: Data Writing. The binary data or FASTA data that the user needs to store is encoded. The encoded DNA sequence is assembled with the designed multilayer address sequence into a plasmid map, which is then synthesized and cloned to form the POP database.

[0075] Step 2: Data Storage. The synthesized POP database is processed through pooling, morphological manipulation, and drying to meet actual storage requirements and ensure long-term storage of DNA information.

[0076] Step 3: Data Retrieval. Based on the user's query request, the database management layer parses the target address; designs the corresponding sgRNA, assembles it with the Cas12a protein into a complex, and specifically cleaves the POP database under suitable temperature and solution conditions to generate sticky ends; enriches the data by ligation through complementary adapters, amplifies the enriched product by PCR, and sequences it; after decoding, the target data is restored.

[0077] Step 4: Data Deletion. Design an sgRNA targeting the address sequence of the data to be deleted, and cut the target plasmid with Cas12a. The uncuttered plasmid and the cut plasmid can be physically separated by gel electrophoresis, thereby realizing the deletion of data and the recovery of undeleted data.

[0078] Step 5: Data Copying. The data to be copied is replicated on a large scale by transforming plasmids into bacteria such as E. coli.

[0079] The data writing in step 1 can be encoded in a way that is compatible with a variety of encoding techniques, including but not limited to modulation coding, yin-yang coding, fountain coding, etc. To obtain a good physical storage pool structure, it is necessary to maintain the consistency of the size of the individual data pool plasmids after block division, and each plasmid contains at least one layer of address sequence.

[0080] The data storage methods in step 2 include, but are not limited to, silica encapsulation, hydrogel microsphere encapsulation, microcapsule sealing, dried DNA, and transfer into live bacterial cells, and are compatible with multiple storage modes.

[0081] In step 3, the data query mode can perform multiple queries according to user needs and database structure, including but not limited to keyword query, Boolean operation logic query, single address query, multi-file query, hierarchical query, etc.

[0082] In step 4, data deletion requires determining the specific database and address information based on the deletion target, and adopting the best sgRNA recognition strategy according to actual needs, including but not limited to single sgRNA recognition, multiple sgRNA recognition, and sgRNA pool recognition with degenerate bases.

[0083] In step 5, the data information copying process can be determined based on user needs and storage status, whether to copy directly or to first use the CORE system to identify and separate valid information before copying.

[0084] The data storage information includes, but is not limited to, any binary information and biological information fragments from nature.

[0085] Example 6: This embodiment provides a plasmid pool (POP) DNA database management system architecture based on the CORE retrieval mechanism, specifically including: Building the POP database: like Figure 4 As shown, taking the storage of poems containing the characters "plum blossom," "orchid," "bamboo," and "chrysanthemum" as an example (abbreviated as "M," "L," "Z," and "J"), the text is converted into binary data. This specifically includes the following steps:

[0086] 1. Data storage and encoding The raw data (such as the poems about "plum blossom", "orchid", "bamboo" and "chrysanthemum") is processed by the database system, converted into DNA sequences through encoding, and assembled into plasmid vectors to complete the physical storage of the data and achieve data preservation.

[0087] 2. Data Retrieval and Backup Retrieval: When a user initiates a query, the system uses the CORE retrieval mechanism to locate the specific plasmid address where the target data is located.

[0088] Backup: To ensure data security, the system can perform backup operations on the stored plasmid library. Backup data can be achieved through transformation and intrahost replication, enabling biological backup and amplification of the plasmid library. Data is read using DNA sequencing.

[0089] In this embodiment, we adopted a modulation coding strategy. First, we mapped every 2 bits of the binary sequence to a base. Then, we randomly selected primers from a four-base modulation primer library with a GC content of 50%, performed an XOR operation with the original sequence, and adjusted the global GC content. Finally, we performed secondary modulation on the possible continuous bases formed locally and recorded the modulation position in the seed sequence.

[0090] The design incorporates a sequence containing dual address information, which is then assembled with the encoded data sequence into four plasmids, each of which is the same size.

[0091] We designed a plasmid pool with two address layers, where the first-level address sequence is a fixed sequence and the second-level address sequence is the address sequence of the "Mei" plasmid. We processed the second-level address sequence and expanded it into three plasmid pools (referred to as "M1", "M2", and "M3") by using a 4-base substitution method, each containing 256 different address sequences.

[0092] The plasmid map was sent to a commercial company for synthesis and transformed into E. coli for cloning, proliferation and preservation, forming the POP database entity.

[0093] Data deletion operation based on CORE: Delete a specified image file from the database.

[0094] The logic management layer parses out the address sequence corresponding to all data blocks in the file.

[0095] For each address sequence, a corresponding sgRNA is designed, which can be used in combination or alone to achieve batch deletion, depending on the actual situation.

[0096] like Figure 5 As shown, the data is stored in a specific POP structure, which contains a plasmid backbone 51 and an inserted data storage fragment 52. The POP database is processed using the cutting step of the CORE retrieval system. Guided by sgRNA, the Cas12a protein precisely cuts the data storage fragment 52 at a specific location on the target plasmid. The target plasmid is targeted and cut, resulting in a linearized plasmid.

[0097] The target DNA was separated by agarose gel electrophoresis. Linearized plasmids and supercoiled closed circular plasmids have different migration rates and can be effectively separated and excised.

[0098] The uncut closed-loop plasmids are recovered, resulting in the POP database after the target files have been deleted.

[0099] CORE-based random retrieval operation: Randomly retrieve a single address and a single data pool from the above database, and combine the sgRNA and the data pool mixture to implement the retrieval logic of "AND", "OR", and "NOT".

[0100] The logic management layer locates the data block where the field is located and its corresponding multi-layer address sequence.

[0101] Design an sgRNA targeting this combined address sequence.

[0102] Incubate the sgRNA with the AmCas12a protein at room temperature for 10 minutes to form an RNP complex.

[0103] Take an appropriate amount of POP database sample and react it with the RNP complex at 37 °C for 5 - 30 minutes. The Cas12a protein completes specific cleavage of the target plasmid.

[0104] At the cleavage site, a tagged Tag (linking tag) is precisely ligated to the end of the data fragment. This ligation reaction has high sequence specificity and can be used as a secondary verification of the target address, greatly reducing the background noise of non-specific retrieval.

[0105] In the figure, 53 represents adding an adapter sequence that is completely complementary to the sticky end generated by Cas12a cleavage, and ligating it at 16 °C using T4 DNA ligase.

[0106] Use the ligation product as a template, perform amplification library construction using universal primers, and output the PCR product.

[0107] When data needs to be read, sequence the PCR product (Sequencing), compare the base sequence obtained by sequencing with the original sequence information, and the sequencing results are as Figures 6a to 6g shown. In fact, various sequencing methods such as high-throughput (NGS) sequencing, nanopore (Nanopore) sequencing, and single molecule real-time sequencing (PacBio) can be used. The meanings of each sequence in the figure are as follows:

[0108] Figure 6a Corresponding to M, that is, "Plum" (Pinyin Mei, English Plum), with the寓意 of "Without experiencing the bitterness of the cold, how can one enjoy the fragrance of the plum blossoms".

[0109] Figure 6b Corresponding to L, that is, "Orchid" (Pinyin Lan, English Orchid), with the寓意 of "The fragrance of the orchid will never change, and the heart will always be like an orchid and never move".

[0110] Figure 6c Corresponding to Z, that is, "Bamboo" (Pinyin Zhu, English Bamboo), with the寓意 of "After countless trials and hardships, it still stands firm, no matter which way the wind blows".

[0111] Figure 6d Corresponding to J, that is, "Ju" (pinyin: Ju, English: Chrysanthemum), it implies "The fragrance permeates Chang'an in the soaring array, and the whole town is clad in golden armor."

[0112] Figure 6e and 6f 6g respectively correspond to mutants of type N1: M-mis1, M-mis2, M-mis3.

[0113] As Figure 7 shown, the complete operation process of database parallel operation in the embodiments of the present invention includes the following two parallel and converging core paths: 1. Data writing and processing path (storage) This path describes the process of storing the original data into the system: Starting point: Data information (original information to be stored).

[0114] Processing: The data first enters the storage engine for encoding and formatting, and the processor completes the calculation and conversion.

[0115] Transmission and storage: The processed data is transmitted to the database management center through the Internet, and the registration, management, and physical (in the form of DNA) storage of the data are completed.

[0116] 2. Data reading and retrieval path (query) This path describes the process of querying and reading data from the system: Starting point: Data node (pointing to the physical location or address where the specific data is stored).

[0117] Retrieval: The node information is input into the CORE retrieval engine, and the engine analyzes the address and starts the biochemical retrieval mechanism.

[0118] Decoding: The retrieved DNA data is converted into digital information through sequencing technology.

[0119] Back transmission: The obtained sequence data is transmitted back to the database management center through the Internet for verification, assembly, and final output.

[0120] Figure 8 Through the encoding structure and mathematical calculation, it intuitively shows how the CORE mechanism realizes the addressing ability of ultra-large-scale data storage. In the embodiments of the present invention, the CORE mechanism significantly improves the database address capacity and the overall capacity.

[0121] 1. Core address structure As shown in the figure, the 16-layer address is composed of 16 independent coding units connected in series horizontally. Each unit (i.e., an "address bit") contains three core parts:

[0122] Figure 8The purple portion represents the nick site: it is 3 base pairs in length and is the site where the CRISPR system performs precise cutting; based on the characteristics of the T4 ligase, there are a total of 16 nick sites.

[0123] Figure 8 The red portion represents the target sequence: it is 20 base pairs (bp) in length and is the key region for the Cas12a system to specifically recognize and bind to it.

[0124] Figure 8 The yellow portion represents the PAM sequence: it is 4 base pairs in length and provides the necessary sequence context for the recognition and initiation of cleavage of the Cas12a protein.

[0125] The standard length of each address bit is 20 + 3 + 4 = 27 bp, so the total length of the entire address sequence is 27 bp × 16 = 432 bp.

[0126] 2. Address capacity and data load Vast Address Space: Since the 20bp target sequence in each address bit can theoretically be designed as any sequence, a single address bit can provide up to 4^20 different sequence combinations, meaning the number of target sequences is 4^20 / 20bp. After concatenating 16 address bits, the system can theoretically generate a total of (4^20)^16 = 2^640 addresses. This constitutes an extremely large logical address space.

[0127] Flexible data load ( Figure 8 (Blue area): Data payload capacity is 0-200kb. Each unique address points to an independent data storage segment. As shown in the blue data payload area on the right side of the figure, each segment can carry 0 to 25,000 bytes of valid data.

[0128] 3. Overall capacity estimation Based on the above design, the theoretical maximum storage capacity of the system can be estimated by the following formula: (data load per address) × (total number of addresses) That is: 200 KB × (4^20)^16 ≈ 45 × 2^570 YB.

[0129] 4. Summary of Technological Advantages This diagram illustrates the core principle of the CORE mechanism's expansion: by decomposing long addresses into multiple short standard units and combining them for encoding, an exponential increase in the number of addresses is achieved within a finite physical DNA length, thus supporting a nearly unlimited theoretical total database capacity far exceeding current needs. This "multi-layer addressing" design is key to overcoming the bottleneck of DNA data storage addressing scale.

[0130] The DNA database management system provided in this invention, through its innovative POP architecture and CORE engine, enables the construction of a large-capacity DNA-based database. This database management system currently supports a 16-layer address structure design, with a maximum capacity of 2... 640 A sequence of addresses, achieving 45×2 570 YB's data storage. This database management system's high density, large capacity, fast retrieval, and parallel scalability make it ideal for scenarios requiring long-term, secure storage of massive amounts of data, such as national archives, scientific data, film and television materials, and medical and health information backups. This system provides a disruptive technological approach with completely independent intellectual property rights to address the data storage challenges of the 1000-byte era, and has broad prospects for industrial application.

[0131] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several equivalent substitutions or obvious modifications can be made without departing from the concept of the present invention, and all such modifications, achieving the same performance or application, should be considered within the scope of protection of the present invention.

Claims

1. A method for managing a plasmid pool DNA database based on CRISPR-Cas12a, characterized in that, Includes the following steps: S1. Data writing steps: Encode the data that the user needs to store, assemble the encoded DNA sequence with the designed multilayer address sequence into a plasmid map, and form a plasmid pool database after synthesis and cloning. S2, Data retrieval steps, specifically including: S21. Based on the user's query request, parse out the target address; S22. Design a single-guide RNA corresponding to the target address, and assemble the single-guide RNA with Cas12a protein into a ribonucleoprotein complex. S23. The ribonucleoprotein complex is used to specifically cut the plasmid pool database under suitable reaction conditions to label the target plasmid; S24. Based on the marker, enrich the dicing product; S25. Amplify and sequence the enriched products, and then decode to restore the target data; S3, Data Deletion Steps, specifically include: S31. Design a single-guide RNA for the address sequence of the data to be deleted; S32. The target plasmid is cleaved by the Cas12a protein; S33. Data deletion is achieved by separating and removing the cleaved plasmids using gel electrophoresis.

2. The method according to claim 1, characterized in that, In step S1, the data types to be stored include binary data information and biological genetic information; the encoding methods include modulation encoding, Huffman encoding, Base64 encoding or fountain code; the construction of the plasmid pool database is based on a hierarchical model, a network model or a relational model.

3. The method according to claim 1, characterized in that, In step S1, the plasmid pool database consists of multiple plasmids, each with a uniform length; each plasmid contains at least one address sequence for addressing.

4. The method according to claim 3, characterized in that, The plasmid includes a fluorescent recognition element or an anti-resistance protein expression element.

5. The method according to claim 1, characterized in that, In step S23, the labeling is achieved by cutting to generate sticky ends and connecting them through a connector complementary to the sticky ends; the suitable reaction conditions include: the reaction buffer contains a reaction solution suitable for the corresponding Cas12a protein and a reagent to inhibit RNA degradation.

6. The method according to claim 1, characterized in that, The Cas12a protein includes AmCas12a, FnCas12a, and LbCas12a variants.

7. The method according to claim 1, characterized in that, In step S2, the data manipulation methods in the data retrieval step include single address access, single file access, batch data access, or logical access.

8. A DNA database management system for implementing the method as described in any one of claims 1 to 9, characterized in that, The system includes: The logic management layer is configured to receive user data manipulation instructions and parse them into unidirectional guide RNA design instructions for specific address sequences. The physical operation layer includes: The plasmid pool database consists of multiple plasmids containing encoded data and multi-layer address sequences; The CRISPR-Cas12a search engine is configured to perform specific cleavage and enrichment operations based on single guide RNA and Cas12a protein.

9. The system according to claim 8, characterized in that, The logical management layer uses an entity-relationship model to abstract and manage the plasmid pool database.

10. The system according to claim 8, characterized in that, The CRISPR-Cas12a search engine includes: The targeted recognition and cleavage module, composed of a ribonucleoprotein complex consisting of a programmable single guide RNA and Cas12a protein, is used to specifically recognize and cleave the address sequence on the target plasmid to generate sticky ends. The specific enrichment module is configured to achieve specific capture and enrichment of target plasmids based on cleavage operations.

Images