Method for stably storing large-scale DNA data

Abstract

The invention discloses a method for stably storing large-scale DNA data, and particularly relates to the field of DNA storage. Comprising the following steps: generating a DNA tag based on a gRNA sequence; introducing the DNA tag into a yeast cell, and culturing into a strain; screening a strain successfully inserted with the DNA tag as a strain containing the DNA tag; performing spore production culture on the strain containing the DNA tag to obtain yeast spores containing the DNA tag; and mixing the yeast spores containing the DNA tags, activated carbon powder and an Arabic gum solution, and drying to obtain a yeast spore library containing the DNA tags. The DNA tag is introduced into yeast, yeast spores with the DNA tag are obtained through culture, the yeast spores with the DNA tag are mixed with activated carbon powder and an Arabic gum solution, coating of the yeast spores with the DNA tag is achieved, and resistance and information stability of the yeast spores with the DNA tag under severe conditions are improved.

Description

Technical Field

[0001] The present application relates to the field of DNA storage, and in particular to a method for stable storage of large-scale DNA data. Background Art

[0002] With the rapid development of information technology and the era of big data, the speed of information generation and communication has increased exponentially. How to efficiently store and manage massive amounts of data has become an urgent problem to be solved today. Existing silicon-based storage technology has gradually become unable to meet the growing demand, especially in terms of storage density, stability and energy consumption. Finding alternative storage media has become a research hotspot. In recent years, deoxyribonucleic acid (DNA) has become one of the first choices for new storage media due to its high storage density, long life and low energy consumption.

[0003] In in vitro DNA storage, DNA molecules are stored in inanimate carriers such as test tubes or silica. Although this method has a certain storage capacity, its long-term stability is limited by changes in the external environment, especially the influence of factors such as temperature, humidity, and oxidation, making it difficult to ensure the integrity and long-term durability of the data. In addition, random reading of in vitro DNA storage is also extremely limited. Reading data usually requires relying on amplification technologies such as polymerase chain reaction (PCR), and PCR can only read and amplify shorter DNA fragments, limiting the practicality of large-scale DNA storage. In addition, existing in vivo DNA storage cannot guarantee the stability and durability of DNA. Summary of the invention

[0004] The main purpose of this application is to provide a method for stable storage of large-scale DNA data, aiming at the fact that existing in vivo DNA storage cannot guarantee the stability and durability of DNA.

[0005] To achieve the above-mentioned objectives, the present application provides a method for stable storage of large-scale DNA data, comprising: generating DNA tags based on gRNA sequences; introducing the DNA tags into yeast cells and culturing them into strains; screening strains with successfully inserted DNA tags from the cultured strains as strains containing DNA tags; spore-forming culture of the strains containing DNA tags to obtain yeast spores containing DNA tags; mixing the yeast spores containing DNA tags, activated carbon powder and gum arabic solution, and drying them to obtain a yeast spore library containing DNA tags.

[0006] Optionally, when the yeast spores containing DNA tags, activated carbon powder and gum arabic solution are mixed, the mass of activated carbon powder added per OD of yeast spores containing DNA tags is 0.33 g, the volume of gum arabic solution is 100 μL, and the volume fraction of gum arabic solution is 15%.

[0007] Alternatively, the DNA tag is introduced into yeast cells using the lithium acetate transformation method of yeast.

[0008] Optionally, the DNA tags comprise at least 200,000.

[0009] Optionally, each DNA tag is a gRNA sequence comprising a first preset number of bases; wherein, the first second preset number of bases of the gRNA sequence have at least three mismatches with the yeast genome sequence, the CG content of the gRNA sequence is 40-60%, the number of consecutive appearances of the same base in the gRNA sequence is less than or equal to a third preset number, the gRNA sequence does not have a hairpin structure, and there are at least two mismatches among the first second preset number of bases of the gRNA sequence.

[0010] Optionally, the first preset number is 20, the second preset number is 12, and the third preset number is 4.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The method for stable storage of large-scale DNA data of the present invention realizes specific labeling of a yeast cell library through large-scale generated DNA tags for storage of large-scale data information; introduces DNA tags into yeast, and cultivates yeast spores with DNA tags to improve the storage stability and durability of DNA; introduces different gRNA "Key" plasmids into a yeast strain library to directly read any DNA, thereby expanding the practicality of large-scale DNA storage; and coats the yeast spores with DNA tags by mixing them with activated carbon powder and gum arabic solution, thereby improving their resistance and information stability under harsh conditions (ultraviolet, high temperature, oxidation). BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 A schematic diagram of a process for stable storage of large-scale DNA data in this application; Figure 2 A flowchart of DNA tag generation in a method for stable storage of large-scale DNA data in this application; Figure 3 A flowchart of obtaining yeast spores containing DNA tags in a method for stable storage of large-scale DNA data in this application; Figure 4 This is a gRNA mismatch test diagram in a method for stable storage of large-scale DNA data in this application; Figure 5 This is a diagram for verifying the specificity of gRNA in a method for stable storage of large-scale DNA data in this application; Figure 6 This is a test diagram of stress resistance in a method for stable storage of large-scale DNA data in this application.

[0013] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical solutions and advantages of this application clearer, the technical solutions in this application will be clearly and completely described below in conjunction with the drawings in this application. Obviously, the described embodiments are part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0015] The first embodiment of the present invention provides a method for stable storage of large-scale DNA data, such as Figure 1 As shown, the specific steps include: Step S1, generating a DNA tag and a tag sequence based on the gRNA sequence; In this embodiment, the DNA tags include at least 200,000, thereby enabling the establishment of a large-scale yeast spore library containing DNA tags. Specifically, each DNA tag is a gRNA sequence containing the 20th base. The method for generating the tag sequence is shown in Figure 2 ; Among them, the first second preset number of bases of the gRNA sequence have at least three mismatches with the yeast genome sequence to ensure that other non-target regions of the genome are not targeted, the CG content of the gRNA sequence is 40-60% to avoid the formation of long homopolymers, the number of consecutive occurrences of the same base in the gRNA sequence is less than or equal to 4, and the gRNA sequence does not have a hairpin structure to ensure the stability and targeting efficiency of the gRNA sequence, and at least two of the first 12 bases of the gRNA sequence are mismatched so that the gRNA expressed by the sequence does not target other tag sequences. Exemplarily, the first preset number is 20, the second preset number is 12, and the third preset number is 4.

[0016] Step S2, such as Figure 3 As shown, all DNA tags are introduced into yeast cells using the lithium acetate transformation method and cultured into strains; further, after obtaining the DNA tags, each DNA tag is combined with ura3 and leu2 respectively, that is, the tag ura3 and the tag leu2 are obtained, and both the tags ura3 and leu2 are introduced into yeast cells.

[0017] Step S3, screening strains with successfully inserted DNA tags from the cultured strains as strains containing DNA tags; Step S4, performing spore culture on the strain containing the DNA tag to obtain yeast spores containing the DNA tag; Step S5, mixing the yeast spores containing the DNA tag, the activated carbon powder and the gum arabic solution, and drying them to obtain a yeast spore library containing the DNA tag; when the yeast spores containing the DNA tag, the activated carbon powder and the gum arabic solution are mixed, the mass of the activated carbon powder added to each OD of the yeast spores containing the DNA tag is 0.33 g, the volume of the gum arabic solution is 100 μL, and the volume fraction of the gum arabic solution is 15%.

[0018] Example 1 Step S1, generating a tag sequence based on the gRNA sequence, i.e. constructing a "Key-Lock" responsive biological element; Step S2, 1 μg of the tag ura3 and the tag leu2 containing the same tag are digested by BsaI, and the tags ura3 and leu2 are introduced into yeast cells expressing Cas9 protein by the yeast lithium acetate transformation method, and then cultured into strains on a solid medium containing nourseothricin and hygromycin B; Step S3, screening the strains into which the DNA tag is successfully inserted as strains containing the DNA tag.

[0019] The strain containing the DNA tag obtained in this example was used to perform a gRNA mismatch test, such as Figure 4 As shown, the details are as follows.

[0020] The gRNA "Key" with different mismatches was introduced into the strain with the corresponding DNA tag using the yeast lithium acetate transformation method, and cultured using CM-Ura-Leu solid medium. The formula of CM-Ura-Leu solid medium is as follows: 6.7g / L amino-free yeast nitrogen base (YNB), 20g / L glucose, 150mg / L threonine, 30mg / L tyrosine, 150mg / L valine, 30mg / L lysine, 100mg / L glutamate, 150mg / L serine, 100mg / L aspartic acid, 50mg / L phenylalanine, 30mg / L isoleucine, 20mg / L arginine, 50mg / L adenine, 100mg / L tryptophan, 20mg / L methionine, 100mg / L histidine, 15g / L agar, pH adjusted to 6.5.

[0021] The results showed that when there was one base mismatch between the gRNA and the DNA tag, the DNA tag was activated and the strain grew on a specific culture medium; when the mismatch reached two or more, the DNA tag remained "locked" and the strain could not grow.

[0022] Example 2 Step S1, such as Figure 5 As shown, 6 generated DNA tags were randomly selected to construct the “Key-Lock” element; Step S2, using BsaI to digest the ura3 and leu2 tags of the six DNA tags, wherein the ura3 and leu2 tags of each DNA tag are 1 μg, and using the yeast lithium acetate transformation method, all the ura3 and leu2 tags are introduced into yeast cells expressing the Cas9 protein, and then cultured into strains on a solid medium containing nourseothricin and hygromycin B; Step S3, screening 6 strains into which the DNA tags are successfully inserted as strains containing DNA tags.

[0023] gRNA specific detection using the strain containing DNA tags obtained in this example The six DNA tags were used to label the strains (accounting for 1 / 10 5 ) were mixed with wild-type strains to form a yeast strain library, and different gRNA "Key" plasmids were introduced using the lithium acetate transformation method of yeast, and cultured using CM-Ura-Leu solid medium. When the proportion of informant strains was as low as 0.001%, the six gRNAs were able to accurately identify the target strains and control their growth on selective medium, proving that the generated gRNA library has good orthogonality and specificity, and can be used for large-scale specific labeling of yeast cells, and then for large-scale data storage.

[0024] Example 3 Step S1, generating a DNA tag and a tag sequence based on the gRNA sequence; Step S2, using the lithium acetate transformation method of yeast, all DNA tags are introduced into yeast cells, and then cultured into strains; further, after obtaining the DNA tags, each DNA tag is combined with ura3 and leu2 respectively, that is, the tag ura3 and the tag leu2 are obtained, and both the tags ura3 and leu2 are introduced into the yeast cells; Step S3, screening the strains into which the DNA tag is successfully inserted as the strains containing the DNA tag; Step S4, shaking culture: the strain containing the DNA tag is cultured in CM-His medium, and the BY4742 strain is cultured in YPD medium for 12 hours; Fusion: Take 1 OD of the cultured strain containing the DNA tag and BY4742 strain, wash twice with ddH2O, mix, add 5 mL of YPD medium, and culture at 30°C for 24 hours; Screening: The bacterial solution was transferred to CM-Met-Lys-His selective medium, the initial OD was set to 0.1, and cultured at 30°C to screen out strains that successfully hybridized and carried DNA tags; Pre-spore production-spore production: transfer the selected strain to GNA medium (pre-spore production medium), with an initial OD of 1, and culture at 30°C until the OD reaches 4-8 (at least doubled twice). Measure the OD every 1 hour after 4 hours of culture; Collect bacteria: Wash with ddH2O 3 times, transfer to spore-forming medium, OD=1, and culture at 25°C for 3-10 days to obtain yeast spore suspension containing DNA tags; The composition of the GNA pre-spore medium is as follows: glucose: 50 g / L; Difco nutrient broth: 30 g / L; yeast extract: 10 g / L. The composition of the spore medium is as follows: potassium acetate: 10 g / L; zinc acetate dihydrate: 0.05 g / L, uracil (Ura): 10 mg / L; leucine (Leu): 20 mg / L; yeast extract: 0.3 g / L; In step S5, 0.15 OD of the yeast spore suspension containing the DNA tag is mixed with 0.05 g of activated carbon powder and 15% gum arabic solution, and then dried in a vacuum rotary evaporator at 30° C. to obtain a yeast spore library containing the DNA tag.

[0025] The unprotected yeast spores (spores) containing DNA tags obtained in step S4 of this example, the yeast spores (encapsulation) containing DNA tags obtained in step S5, and the vegetative yeast cell bank were exposed to the following conditions: 254 nm ultraviolet irradiation and 60° C. high temperature treatment. The bacterial liquid was spread on CM-His solid culture medium for counting culture. The formula is as follows: amino-free yeast nitrogen source (YNB): 6.7g / L; glucose: 20g / L; threonine (Thr): 150mg / L; tyrosine (Tyr): 30mg / L; valine (Val): 150mg / L; lysine (Lys): 30mg / L; glutamic acid (Glu): 100mg / L; serine (Ser): 150mg / L; aspartic acid (Asp): 100mg / L; phenylalanine (Phe): 50mg / L; isoleucine (Ile): 30mg / L; arginine (Arg): 20mg / L; adenine (Ade): 50mg / L; tryptophan (Trp): 100mg / L; uracil (Ura): 50mg / L; leucine (Leu): 100mg / L; adjust pH to 6.5.

[0026] The survival rate of cells was evaluated for stress resistance by the above method. The evaluation results are as follows: Figure 6 As shown in the figure, it can be seen that the yeast spores containing DNA tags obtained in step S5 remain highly stable under harsh environmental conditions such as 254 nm ultraviolet band irradiation and 60°C high temperature treatment, and are suitable for long-term storage needs.

[0027] The above are only preferred embodiments of the present application, and are not intended to limit the patent scope of the present application. Any equivalent structure or equivalent process transformation made using the contents of the present application specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. A method for stable storage of large-scale DNA data, characterized in that: include: Generate DNA tags based on gRNA sequences; Introducing the DNA tag into yeast cells and culturing them into strains; From the cultured strains, strains into which the DNA tag is successfully inserted are selected as strains containing the DNA tag; Performing spore culture on the strain containing the DNA tag to obtain yeast spores containing the DNA tag; The yeast spores containing the DNA tag, activated carbon powder and gum arabic solution are mixed and dried to obtain a yeast spore library containing the DNA tag.

2. The method for stable storage of large-scale DNA data according to claim 1, characterized in that: When the yeast spores containing DNA tags, activated carbon powder and gum arabic solution are mixed, the mass of activated carbon powder added to each OD of yeast spores containing DNA tags is 0.33 g, the volume of gum arabic solution is 100 μL, and the volume fraction of gum arabic solution is 15%.

3. The method for stable storage of large-scale DNA data according to claim 1, characterized in that: The DNA tag is introduced into yeast cells by the lithium acetate transformation method of yeast.

4. The method for stable storage of large-scale DNA data according to claim 1, characterized in that: The DNA tags include at least 200,000.

5. The method for stable storage of large-scale DNA data according to claim 4, characterized in that: Each of the DNA tags is a gRNA sequence comprising a first preset number of bases; Among them, the first second preset number of bases of the gRNA sequence have at least three mismatches with the yeast genome sequence, the CG content of the gRNA sequence is 40-60%, the number of consecutive appearances of the same base in the gRNA sequence is less than or equal to a third preset number, the gRNA sequence does not have a hairpin structure, and at least two of the first second preset number of bases of the gRNA sequence are mismatched.

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