SgRNA and CRISPR / Cas9 gene editing system targeting TATA-box and ATG of penaeus japonicus and application

Abstract

The invention discloses an SgRNA and CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9 gene editing system for targeting TATA-box and ATG (adenosine triphosphate) of penaeus japonicus and application of the system. The sequence of the SgRNA is as shown in SEQ ID No.1 to SEQ ID No.26, and the SgRNA can target TATA-box and ATG (adenosine triphosphate) of penaeus japonicus. According to the invention, a mixture of SgRNA and Cas9 protein is injected into fertilized eggs of penaeus japonicus so as to obtain random mutants of penaeus japonicus, and indel mutation is generated for screening. The method is helpful for constructing a penaeus japonicus random mutant library with rich genetic variation, is also helpful for providing important technical support for precise breeding of the penaeus japonicus, and lays a foundation for improved variety breeding of the penaeus japonicus.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene editing, and in particular relates to an SgRNA and CRISPR / Cas9 gene editing system and application targeting TATA-box and ATG of Japanese shrimp. Background Art

[0002] Japanese shrimp ( Marsupenaeus japonicus ) Formerly known as Japanese capsule shrimp, it belongs to the order Decapoda, family Penaeidae, and genus Penaeus. It is distributed in the tropical waters of the Indian-Western Pacific, the east coast of Africa, Malaysia, Japan, North Korea, and the southeastern coast of China. It is one of the main shrimp farming varieties in China. Japanese shrimp grows rapidly, tolerates drought and exposure, has bright colors and high economic value. It is very popular in the aquatic product market and farmers. It is farmed in coastal provinces and cities from the south to the north of my country and has formed a certain scale. At present, there is only one new variety of Japanese shrimp in China-"Minhai No. 1", which is bred based on traditional selective breeding methods. Due to the low heritability of stress resistance and disease resistance and the difficulty in measuring, traditional breeding has certain limitations. Molecular breeding is the most effective way to break the bottleneck of breeding. Therefore, breaking through the molecular breeding technology of Japanese shrimp is an important way to create improved varieties.

[0003] CRISPR / Cas9 gene editing technology can delete or insert DNA sequences at the DNA level. At present, this technology is almost always used for functional knockout of single known genes in crop and some aquatic animal genetic breeding, but there are no reports on the establishment of a large-scale random mutant library for shrimp based on this technology. The establishment and development of a CRISPR / Cas9-based random mutant library in Japanese shrimp will, on the one hand, help to construct a Japanese shrimp random mutant library with rich genetic variation, and on the other hand, provide important technical support for the precision breeding of shrimp, laying the foundation for the breeding of Japanese shrimp varieties. Summary of the invention

[0004] The purpose of the present invention is to provide a SgRNA targeting TATA-box and ATG of Japanese shrimp and a CRISPR / Cas9 gene editing system and application. The SgRNA can target the promoter region TATA-box and the start codon ATG of Japanese shrimp, thereby achieving indel mutation of random mutation of Japanese shrimp.

[0005] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions: The present invention provides an SgRNA targeting Japanese shrimp TATA-box and ATG, and the sequence of the SgRNA is shown as SEQ ID No.1 to SEQ ID No.26.

[0006] The present invention also provides a CRISPR / Cas9 gene editing system, which comprises the SgRNA sequence and the Cas9 protein.

[0007] The present invention also provides a vector comprising the nucleotide sequence of the SgRNA.

[0008] Furthermore, the vector is a pT7-gRNA vector.

[0009] The present invention also provides amplification primers for the SgRNA targeting TATA-box and ATG of Japanese shrimp, and the sequences of the primers are shown in SEQ ID No.27 to SEQ ID No.53.

[0010] The present invention also provides a kit for constructing a Japanese shrimp mutant, which comprises the SgRNA and Cas9 protein.

[0011] The present invention also provides an application of the CRISPR / Cas9 gene editing system in editing the DNA of Japanese shrimp, and the application steps are: injecting the mixture of the SgRNA and the Cas9 protein into the fertilized eggs of Japanese shrimp, extracting the genomic DNA from the injected individuals after cultivation for genome resequencing, selecting individuals with indel mutations, and obtaining Japanese shrimp mutants.

[0012] Furthermore, the mixing mass ratio of the SgRNA and Cas9 protein is 1:1~2.

[0013] Furthermore, the concentration of the SgRNA is 100 ng / μL~150 ng / μL; the concentration of the Cas9 protein is 200 ng / μL~250 ng / μL.

[0014] The present invention also provides the use of the SgRNA, the vector or the CRISPR / Cas9 gene editing system in constructing a Japanese shrimp random mutant library.

[0015] The present invention also provides the use of the SgRNA, the vector or the CRISPR / Cas9 gene editing system in the breeding of Japanese shrimp varieties.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects: According to the target design principle of SgRNA, the present invention designs a knockout target site for the promoter region TATA-box and the start codon ATG of Japanese shrimp, obtains SgRNA that can target Japanese shrimp TATA-box and ATG, and co-injects it with Cas9 protein into the fertilized eggs of Japanese shrimp to obtain random mutants of Japanese shrimp, which have indel mutations and are screened. This helps to construct a Japanese shrimp random mutant library with rich genetic variation, and also helps to provide important technical support for the precise breeding of shrimp, laying the foundation for the breeding of Japanese shrimp. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Design and synthesis diagram of SgRNA targeting TATA-box and ATG of Japanese shrimp; Figure 2 This is a picture of microinjection of fertilized eggs of Japanese shrimp; Figure 3 is the proportion of individual Indels in different groups; Figure 4 These are the indel mutation types that appear in different regions after injection, where A is the mutation type near the TATA-box, and B is the mutation type near the ATG. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described in detail with reference to the following specific examples.

[0019] In the following examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0020] Example 1 1. Experimental Materials The Japanese shrimp broodstock used in the present invention are from Rizhao Ocean and Fishery Research Institute, 280 days old, weighing about 220g. Rearing conditions: natural seawater, 18°C, temporarily raised for 10 days, continuously aerated, regularly fed with sterilized sandworms every day, and changed water to clean.

[0021] 2. Synthesis of SgRNA editing target site The specific operation steps are as follows: According to the target design principle of SgRNA, the knockout target site is designed for the TATA-box and the start codon ATG in the promoter region of Japanese shrimp (such as Figure 1 Primers were designed according to the pT7-gRNA plasmid sequence and the target site. The sequences of SgRNA are shown in SEQ ID No. 1 to SEQ ID No. 26, and the primer sequences for amplifying SgRNA are shown in Table 1.

[0022] SgRNA1:aatTATAcAcaaaaaaaaaaa(SEQ ID No.1) SgRNA2:aataTATAcAcaaaaaaaaa(SEQ ID No.2) SgRNA3:fatherTATAcAcaaaaaaaaa(SEQ ID No.3) SgRNA4:aataaaTATAcAcaaaaaaa(SEQ ID No.4) SgRNA5:aataaaaTATAcAcaaaaaa(SEQ ID No.5) SgRNA6:aataaaaaTATAcAcaaaaa(SEQ ID No.6) SgRNA7:aataaaaaaTATAcAcaaaa(SEQ ID No.7) SgRNA8:aataaaaaaaTATAcAcaaa(SEQ ID No.8) SgRNA9:aataaaaaaaaTATAcAcaa(SEQ ID No.9) SgRNA10:aataaaaaaaaaTATAcAca(SEQ ID No.10) SgRNA11:aataaaaaaaaaaTATAcAc(SEQ ID No.11) SgRNA12:aatATGaaaaaaaaaaaaaaa(SEQ ID No.12) SgRNA13:aataATGaaaaaaaaaaaaa(SEQ ID No.13) SgRNA14:aataaATGaaaaaaaaaaaaa(SEQ ID No.14) SgRNA15:aataaATGaaaaaaaaaaa(SEQ ID No.15) SgRNA16:aataaaaATGaaaaaaaaaaa(SEQ ID No.16) SgRNA17:aataaaaaaATGaaaaaaaaa(SEQ ID No.17) SgRNA18:aataaaaaaATGaaaaaaaaa(SEQ ID No.18) SgRNA19: aataaaaaaaATGaaaaaaa (SEQ ID No. 19); SgRNA20: aataaaaaaaaATGaaaaaa (SEQ ID No. 20); SgRNA21: aataaaaaaaaaATGaaaaa (SEQ ID No. 21); SgRNA22: aataaaaaaaaaaATGaaaa (SEQ ID No. 22); SgRNA23: aataaaaaaaaaaaATGaaa (SEQ ID No. 23); SgRNA24: aataaaaaaaaaaaaATGaa (SEQ ID No. 24); SgRNA25: aataaaaaaaaaaaaaATGa (SEQ ID No. 25); SgRNA26: aataaaaaaaaaaaaaaATG (SEQ ID No. 26).

[0023] Table 1 Primer sequences

[0024] Note: Lowercase letters represent substituted bases: a represents any base, t represents A or G, and c represents A or T.

[0025] Using pT7-gRNA plasmid as template, amplification was performed according to the following system:

[0026] Amplification conditions were: 98°C for 3 min; (94°C, 30 s; 55°C, 30 s; 68°C, 20 s) × 30 cycles; 68°C for 7 min. The resulting PCR product was purified, recovered, and stored at -20°C.

[0027] The PCR product obtained in the previous step was used as a template and in vitro transcription was performed using a T7 in vitro transcription kit. The specific transcription system is as follows:

[0028] Incubate at 37°C for 4 h and purify SgRNA using RNeasy Mini kit.

[0029] 3. Microinjection Sample Preparation The purified SgRNA was measured for concentration and quality using a Nanodrop micro-spectrophotometer and 1.5% agarose electrophoresis. The 26 SgRNAs were diluted to 100 ng / μL and mixed in equal volumes for later use; the commercially available Cas9 protein (Invitrogen) was diluted with enzyme-free water at 200 ng / μL for later use. The previously mixed SgRNA and the diluted Cas9 protein were then prepared into the injection sample in a 1:1 volume ratio.

[0030] 4. Treatment of Japanese shrimp broodstock Select female shrimps with well-developed ovaries, remove the unilateral eyestalk, and disinfect them. Then raise them at 18℃ with continuous aeration and feed them with disinfected sandworms at 4 pm every day.

[0031] 5. Microinjection of one-cell embryos of Penaeus japonicus The day before injection, grooves were made using a mold and 1.5% agarose and cooled for later use. Seawater with a salinity of 30‰ was prepared and sterilized by filtration using a 0.22 μm filter membrane.

[0032] Obtaining the first-cell stage fertilized eggs of Japanese shrimp: The Japanese shrimp broodstock are monitored in real time from 19:00 to 2:00 in the morning by taking turns on duty and real-time observation, so as to obtain the newly discharged Japanese shrimp fertilized eggs. When the Japanese shrimp begins to lay eggs, the fertilized eggs are filtered with a 200-mesh silk sieve as soon as possible. Under a stereo microscope, the fertilized eggs are gently arranged in the agarose grooves with sterile seawater. The pneumatic picoliter pump is used to inject the fertilized eggs one by one ( Figure 2 The injected embryos were left to rest for 2 h in aerated seawater with a salinity of 30‰ and a culture temperature of 22˚C, and dead embryos were picked out at any time.

[0033] 6. Detect the mutation of the sample after injection 60 days after injection, the injected individuals were taken and genomic DNA was extracted according to the "Animal Tissue Genome Extraction Kit" of Kangwei Century Company. The control group was individuals that were UV-irradiated at the embryonic stage and the ordinary control group that was not treated. The DNA samples were then mailed to a sequencing company for genome resequencing, and the indel mutations of random mutants were analyzed at the whole genome level.

[0034] The identification results of Indel distribution are as follows Figure 3 As shown, microinjection group > ultraviolet irradiation group > normal control group.

[0035] The types of indel mutations in different regions after injection are as follows Figure 4 As shown, A is the mutation type near the TATA-box, and B is the mutation type near the ATG.

[0036] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. An SgRNA targeting TATA-box and ATG of Japanese shrimp, characterized in that The sequence of the SgRNA is shown in SEQ ID No.1 to SEQ ID No.

26.

2. A CRISPR / Cas9 gene editing system, characterized in that: It comprises the SgRNA sequence and Cas9 protein described in claim 1.

3. A carrier, characterized in that The vector comprises the SgRNA of claim 1.

4. The amplification primers for the SgRNA targeting TATA-box and ATG of Japanese shrimp according to claim 1, characterized in that The sequences of the primers are shown in SEQ ID No.27 to SEQ ID No.

53.

5. Use of the sgRNA described in claim 1 or the CRISPR / Cas9 gene editing system described in claim 2 in editing Japanese shrimp DNA.

6. The use according to claim 5, characterized in that: The application method is: injecting the mixture of SgRNA and Cas9 protein described in claim 1 into the fertilized eggs of Japanese shrimp, extracting genomic DNA from the injected individuals after cultivation for genome resequencing, selecting individuals with indel mutations, and obtaining Japanese shrimp mutants.

7. The method according to claim 6, characterized in that The mixing mass ratio of the SgRNA and Cas9 protein is 1:1~2.

8. The method according to claim 6, characterized in that The concentration of the SgRNA is 100 ng / μL~150 ng / μL; the concentration of the Cas9 protein is 200 ng / μL~250 ng / μL.

9. Use of the sgRNA described in claim 1 or the CRISPR / Cas9 gene editing system described in claim 2 in constructing a Japanese shrimp mutant library.

10. Use of the SgRNA described in claim 1 or the CRISPR / Cas9 gene editing system described in claim 2 in the breeding of Japanese shrimp varieties.

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