Preparation and purification method of low-toxicity low-sensitivity high-purity ssDNA for injection
Through seamless connection and efficient nucleic acid purification technology, the toxicity problem of traditional ssDNA purification methods on the fertilized egg link is solved, and high-purity, low-toxicity and high-efficiency ssDNA preparation and purification are achieved, significantly improving the efficiency of gene editing and egg survival rate.
Patent Information
- Application Number
- CN202411632471.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-15
- Publication Date
- 2025-03-11
AI Technical Summary
In the prior art, the purification methods of ssDNA are mostly purified by gel cutting and recycling, which leads to excessive toxicity on the downstream delivery of fertilized eggs and greatly reduced the effect.
The target fragment was constructed into the pRB322 skeleton by seamless ligation, PCR identification and Sanger sequencing identification were performed, digested and electrophoretic, and purified by Lamda exonuclease, and further dialysis and lyophilization were obtained with high purity ssDNA.
The preparation and purification of ssDNA with low toxicity, low sensitivity and high purity was achieved, which improved the egg survival rate and gene editing efficiency of injection, and the highest positive rate reached 52%.
Smart Images
Figure HDA0005136278250000011
Abstract
Description
Technical Field
[0001] The invention relates to the biological field, and in particular to a method for preparing and purifying low-toxic, low-sensitivity and high-purity ssDNA for injection. Background Art
[0002] With the rapid development of gene editing technology, improving gene editing efficiency has become a research focus of major industries. CRISPR / Cas9 technology has been highly valued by scientific researchers since 2013 and has become the main research method for gene knockout and gene knock-in. However, when knocking in large fragments, traditional double-stranded DNA (dsDNA) donors have problems of random insertion and low knock-in efficiency, which limits its application in precise gene editing. In contrast, long single-stranded DNA (Long ssDNA) stands out for its advantages such as high knock-in efficiency and ability to effectively avoid the problem of random insertion of dsDNA fragments in the genome. Studies have shown that when Long ssDNA is used as a "Donor template", the fragment knock-in efficiency can reach 8.5-100%, greatly shortening the modeling cycle. However, the purification method of ssDNA in the prior art is mostly purified by tapping and recovery, which causes excessive toxicity to the downstream delivery of fertilized eggs, resulting in greatly reduced effects. Summary of the invention
[0003] The purpose of the present invention is to provide a method for preparing and purifying ssDNA for injection with low toxicity, low sensitivity and high purity. The present invention achieves the above purpose through the following technical scheme: a method for preparing and purifying ssDNA for injection with low toxicity, low sensitivity and high purity, amplifying the target fragment, and constructing the target fragment into the skeleton through seamless connection;
[0004] After the positive clones were obtained by PCR identification, they were identified by Sanger sequencing and selected as positive templates.
[0005] Perform PCR amplification on the target region, and clean the amplified product using a cleaning kit;
[0006] The product was digested with Lamda exonuclease for a certain time and temperature, and the digested product was run on gel electrophoresis;
[0007] Place the product from the previous step into an electrophoresis tank for electrophoresis, and run all the target single-stranded DNA in the gel block into the liquid. Remove the gel block, seal the dialysis bag, and place it in a beaker containing ddH2O and a rotor for rotation and purification for a certain period of time to remove the salt ions in the solution.
[0008] The purified liquid was aspirated into a centrifuge tube and placed in a freeze dryer for freeze drying for later use.
[0009] Furthermore, the backbone is the pRB322 backbone.
[0010] Furthermore, the digestion temperature is about 37° C., and the digestion time is about 30 minutes.
[0011] Furthermore, the purification time was approximately 16 hours.
[0012] Furthermore, the target region was amplified by PCR using a 5-terminal phosphorylated primer.
[0013] Compared with the prior art, the low-toxic, low-sensitivity, high-purity ssDNA preparation and purification method for injection of the present invention has the following beneficial effects: a major breakthrough in the survival rate of injected eggs and gene editing efficiency, with the highest positive rate reaching 52%. Compared with traditional preparation and purification methods, this method causes less damage to eggs and has higher sample purity. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 It is a schematic diagram comparing the survival rate and positive rate of various types of injection materials of the present invention. DETAILED DESCRIPTION
[0015] A method for preparing and purifying low-toxic, low-sensitivity, high-purity ssDNA for injection comprises the following steps:
[0016] 1) Amplify the target fragment and construct it into the pRB322 backbone by seamless ligation.
[0017] 2) After the positive clones were obtained by PCR, they were identified by Sanger sequencing and selected as positive templates.
[0018] 3) Use the 5-terminal phosphorylated primer to perform PCR amplification on the target region, and use a cleaning kit to clean the amplified product.
[0019] 4) Digest the product with Lamda exonuclease and incubate at 37°C for about 30 min. Run the digested product on gel electrophoresis and cut the digested single-stranded band to avoid interference from other miscellaneous bands and uncut double-strands.
[0020] 5) Place the cut strips into a 14 kDa dialysis bag and add 3 ml of clean TAE solution, squeeze out all the air, keep as little liquid as possible, 1-2 ml, and clamp both ends with dialysis clips.
[0021] 6) Place the product from the previous step into an electrophoresis tank for electrophoresis, and run all the target single-stranded DNA in the gel block into the liquid. Remove the gel block, seal the dialysis bag, and place it in a beaker containing ddH2O and a rotor for rotation and purification for about 16 hours to remove the salt ions in the solution.
[0022] 7) The purified liquid is aspirated into a centrifuge tube and placed in a freeze dryer for freeze drying.
[0023] For a comparison of the survival rate and positive rate of various types of injection materials in the present invention, please refer to Figure 1 .
[0024] The present invention has made great breakthroughs in the survival rate of injected eggs and gene editing efficiency, with the highest positive rate reaching 52%. Compared with traditional preparation and purification methods, this method causes less damage to eggs and has higher sample purity.
[0025] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any figure mark in the claims should not be regarded as limiting the claims involved.
[0026] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.
Claims
1. A method for preparing and purifying low-toxic, low-sensitivity, high-purity ssDNA for injection, characterized by: The following steps are involved: Amplify the target fragment and build it into the skeleton through seamless connection; After the positive clones were obtained by PCR identification, they were identified by Sanger sequencing and selected as positive templates. Perform PCR amplification on the target region, and clean the amplified product using a cleaning kit; The product was digested with Lamda exonuclease for a certain time and temperature, and the digested product was run on gel electrophoresis; Place the product from the previous step into an electrophoresis tank for electrophoresis, and run all the target single-stranded DNA in the gel block into the liquid. Remove the gel block, seal the dialysis bag, and place it in a beaker containing ddH2O and a rotor for rotation and purification for a certain period of time to remove the salt ions in the solution. The purified liquid was aspirated into a centrifuge tube and placed in a freeze dryer for freeze drying for later use.
2. The method for preparing and purifying low-toxic, low-sensitivity, high-purity ssDNA for injection according to claim 1, characterized in that: The backbone is the pRB322 backbone.
3. The method for preparing and purifying low-toxic, low-sensitivity, high-purity ssDNA for injection according to claim 1, characterized in that: The digestion temperature is about 37° C., and the digestion time is about 30 min.
4. The method for preparing and purifying low-toxic, low-sensitivity, high-purity ssDNA for injection according to claim 1, characterized in that: The purification time was approximately 16 hours.
5. The method for preparing and purifying low-toxic, low-sensitivity, high-purity ssDNA for injection according to claim 1, characterized in that: The target region was amplified by PCR using a 5-terminal phosphorylated primer.