Method for targeted creation of fast-growing new tilapia strain

Abstract

The invention discloses a method for targeted creation of a fast-growing new tilapia strain, and belongs to the technical field of fish cultivation. The method specifically comprises the following steps: (1) taking a fragment, as shown in SEQ ID NO.1, on a third exon of a pomc gene as a gRNA target sequence; (2) designing and synthesizing a gRNA primer sequence; (3) amplifying by adopting the gRNA primer sequence and the gRNA plasmid, and then transcribing and purifying to obtain a gRNA fragment; (4) transferring the gRNA fragments into fertilized eggs of tilapia mossambica through gene editing to obtain F0-generation positive fish; and (5) hybridizing the F0-generation positive male fish with the wild-type female fish, screening out F1-generation parent fish, and culturing the F1-generation parent fish to obtain F2-generation tilapia with homozygous deletion mutation. According to the invention, the pomc homozygous knockout line is established in the Nile tilapia mossambica for the first time, the new fast-growing line is a complete gene function deletion model, the cost is lower, the efficiency is higher, and the phenotype can be stably inherited.

Description

Technical Field

[0001] The invention belongs to the technical field of fish breeding, and in particular relates to a method for creating a new fast-growing tilapia strain in a targeted manner. Background Art

[0002] The excellent traits of fish are closely related to the economic benefits of aquaculture. In order to further increase the output of aquatic products in my country, my country has made rapid progress in breeding technology research and development and cultivation of excellent varieties in recent years.

[0003] Hybridization and selective breeding are the main methods of fish breeding. However, the breeding cycle of hybridization is often long, and due to the existence of reproductive isolation between species, it is difficult to form fertile strains through distant hybridization. Therefore, hybrid breeding has many limitations. Gene editing breeding has a clear purpose and is more efficient and rapid. Gene editing often targets a specific gene, has tissue-specific effects on function, and often has no effect on fertility, which is conducive to the stable inheritance of new phenotypes.

[0004] In the arcuate nucleus, there are two types of neurons that are particularly important for controlling appetite, namely pro-opiomelanocortin (POMC) neurons and agouti-related peptide (AgRP) neurons. POMC neurons promote satiety, while AgRP neurons promote hunger. POMC neurons produce POMC protein, which is a precursor of proteins such as α-melanocyte stimulating hormone (α-MSH), endorphins, and adrenocorticotropic hormone (ACTH). Among them, α-MSH is an important activating ligand of melanocortin receptor-4 (MC4R) and plays a central role in weight regulation (Erica JP Anderson et al. Regulation of feeding and energy homeostasis by α-MSH. J Mol Endocrinol. 2016 May; 56 (4): T157-74. doi: 10.1530 / JME-16-0014). Injection of a synthetic α-MSH antagonist can increase food intake (Frank H. Koegler et al. Central melanocortin receptors mediate changes in food intake in the rhesus macaque. 2001 Jun; 142(6): 2586-92. doi: 10.1210 / endo.142.6.8198.).

[0005] Existing studies have shown that a POMC mutant was constructed in mice using a targeted plasmid, and it was found that the absence of POMC in mice would lead to obesity (Linda Yaswen et al. Obesity in the mouse model of pro-opiomelanocortin deficiency responds to peripheral melanocortin. Nat Med. 1999 Sep; 5(9): 1066-70. doi: 10.1038 / 12506.). Conditional knockout of important genes such as Tsc1 and Manf in POMC neurons in mice will lead to obesity (Qin Tang et al. MANF in POMC Neurons Promotes Brown Adipose Tissue Thermogenesis and Protects Against Diet-Induced Obesity. Diabetes. 2022 Aug; 71(11): 2344-2359. doi: 10.2337 / db21-1128; Hiroyuki Morie et al. Critical role for hypothalamic mTOR activity in energy balance. Cell Metab. 2009 Apr; 9(4): 362-74. doi: 10.1016 / j.cmet.2009.03.005.), but except for a few model species, there is no mature conditional knockout technology in other species such as fish. In addition, after the injection of pomc siRNA into the brain ventricle of mandarin fish, the feeding amount and domestication rate of mandarin fish can be increased (Lu Hailin. Study on the effect of proopiomelanocortin on mandarin fish domestication and its regulatory pathway. Huazhong Agricultural University. June 2021). Although the above two methods can simply and conveniently silence the function of POMC, there are still problems of limited efficiency and instability of inheritance. Therefore, how to stably and efficiently knock out the pomc gene in fish still needs further exploration. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for creating a new fast-growing tilapia strain in a targeted manner, which can efficiently and stably knock out the pomc gene without affecting other genes.

[0007] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0008] The object of the present invention is to provide a method for creating a new fast-growing tilapia strain in a targeted manner, comprising the following steps:

[0009] (1) The fragment of the third exon of the pomc gene as shown in SEQ ID NO.1 was used as the gRNA target sequence;

[0010] (2) gRNA primer sequences as shown in SEQ ID NOs. 2 and 3 were designed and synthesized according to the gRNA target sequence;

[0011] (3) using gRNA primer sequence and gRNA plasmid amplification and then transcription and purification to obtain gRNA fragments;

[0012] (4) transferring the gRNA fragment into the fertilized eggs of tilapia by gene editing, and obtaining F0 generation positive fish by primer detection as shown in SEQ ID NOs. 4 and 5 after incubation;

[0013] (5) The positive male fish of the F0 generation are hybridized with the wild-type female fish to select the F1 generation broodstock, and the F1 generation broodstock are raised to sexual maturity and then mated to obtain the F2 generation tilapia with the homozygous deletion mutation.

[0014] Furthermore, the amplification reaction system in step (3) is as follows: 2×Taq Master Mix 25 μL, gRNA F / R 1 μL, gRNA plasmid 0.3 μL, and finally filled to 50 μL with RNase-free water.

[0015] Furthermore, the reaction conditions in step (3) are: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 15 s, and 38 cycles; 72°C for 10 min, and 12°C for 10 min.

[0016] Furthermore, the transcription system in step (3) includes: 1 μL of T7 / SP6 RNA polymerase, 2 μL of 10×Transcription buffer, 2 μL of 10×rNTP Mix, X μL of RNase inhibitor, and (14-x) μL of RNase-free water.

[0017] Furthermore, in step (4), the Cas9 protein and gRNA are mixed and then injected into tilapia fertilized eggs.

[0018] Furthermore, the concentrations of Cas9 protein and gRNA were both 800-1000 ng / μL, and the mixed volume ratio of the two was 1:1.

[0019] Furthermore, the target fragment amplified by the primer in step (4) is 133 bp. When a large number of new unknown bands and heteroduplexes appear in the test object compared with the control group, it is judged to be a mutant F0 generation positive fish.

[0020] Furthermore, in step (5), when screening F1 generation broodstock, male and female tilapia with frameshift mutations of the same mutation type are used as F1 generation broodstock.

[0021] Another object of the present invention is to provide a use of the pomc gene knockout tilapia mutant obtained by the above method in constructing an animal model related to pomc gene knockout syndrome or in drug screening.

[0022] Beneficial effects of the present invention:

[0023] The present invention mutates the pomc gene of tilapia by using CRISPR / Cas technology, and finds that pomc knockout significantly improves the growth rate 40 days and 90 days after hatching. At the same time, compared with the previous gene knockout technology and knockdown, it has the characteristics of low cost, low toxicity, high accuracy, high efficiency, easy operation, etc., and the obtained phenotype can be stably inherited, which is conducive to the cultivation of new strains.

[0024] The present invention designs a unique sgRNA sequence on the pomc gene, which can not only quickly and efficiently knock out the pomc gene in tilapia, but also will not affect other genes, thus forming a tilapia with specific knockout of the pomc gene.

[0025] The present invention successfully established a pomc homozygous knockout line in Nile tilapia for the first time through CRISPR / Cas9 gene editing technology. This new fast-growing strain is a complete gene function loss model with lower cost and higher efficiency, and the fast-growing phenotype can be stably inherited, and its excellent traits can be maintained without any further operation. After 40 days of mixed breeding after hatching, it was found that the weight and body length of the tilapia pomc homozygous mutant were significantly higher than those of the wild type and heterozygous mutant. At the same time, it was also found that the weight, body length, body width and body thickness of the pomc homozygous mutant were also significantly greater than those of the wild type 90 days after hatching. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The establishment of pomc homozygous knockout line of Nile tilapia; A) pomc gene structure and mutation detection; the target site is on the third exon, and the Sanger sequencing results show that there are different types of mutations, the PAM region is marked with red fonts, and the missing bases are marked with (-); B) Construction of pomc homozygous mutant line, wild type, heterozygous mutation and homozygous mutation are represented by "+ / +", "+ / -" and "- / -", respectively; C) PAGE electrophoresis detection of pomc homozygous mutant; D) Sanger sequencing verification of pomc homozygous mutant; E) Schematic diagram of POMC and feeding regulation pathway;

[0027] Figure 2 For 40dpf pomc - / - ,pomc + / -And the box plot of the growth experiment of wild-type (WT) tilapia; A) Body weight; B) Body length; Statistical analysis was performed using unpaired two-tailed Student's t-test, ***, P<0.001; NS, no significant difference;

[0028] Figure 3 For 90dpf pomc - / - Box plot of growth experiment of tilapia (A) and wild type (WT); A) body weight; B) body length; C) body width; D) body height; Statistical analysis was performed using unpaired two-tailed Student's t-test, ***, P<0.001. DETAILED DESCRIPTION

[0029] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0030] Example 1 Effective targeting of the pomc gene of Nile tilapia using CRISPR / Cas9 gene editing technology

[0031] 1. Target design

[0032] The full sequence of the tilapia pomc gene was downloaded from NCBI, and a target site was designed on the third exon of pomc according to the CRISPR / gRNA target sequence design website (http: / / zifit.partners.org / ZiFiT / ). The target site sequence was: 5'-CCAGCTCTGTCACTCTGACCTCAC-3' (SEQ ID NO.1). After the gRNA primer sequence was designed, it was sent to Qingke Company for synthesis.

[0033] pomc-gRNA-F: 5'-TAATACGACTCACTATAGGCCTGAGAAGAAGGTGAGGGTTTTAGAGCTAGAAATAGC-3'; (SEQ ID NO. 2)

[0034] pomc-gRNA-R: 5'-AGCACCGACTCGGTGCCAC-3'. (SEQ ID NO.3)

[0035] 2. gRNA and Cas9 synthesis and microinjection

[0036] (1) gRNA synthesis

[0037] 1) Using gRNA plasmid (Minghui Li, et al. Efficient and heritable gene targeting in tilapia by CRISPR / Cas9. Genetics. 2014 Jun; 197(2): 591-9. doi: 10.1534 / genetics.114.163667.) as template, amplify and recover the fragments with the designed F primer and universal R primer, and Taq DNA polymerase (2×Taq Master Mix) was purchased from Takara (Takara, China). The reaction system is shown in Table 1.

[0038] Table 1 Reaction system

[0039]

[0040] Reaction conditions:

[0041] 95℃ for 3min; 95℃ for 30s, 60℃ for 30s, 72℃ for 15s, 38 cycles; 72℃ for 10min, 12℃ for 10min.

[0042] 2) In vitro transcription

[0043] In vitro mRNA transcription kit T7 mMESSAGE The kit was purchased from Ambion (Ambion, USA), and in vitro transcription was performed using T7 RNA polymerase. The system is shown in Table 2.

[0044] Table 2 In vitro transcription reaction system

[0045]

[0046] After mixing, place in a water bath at 37℃ for 2h. Add 2μL of DNase I and place in a water bath at 37℃ for 15 minutes. Add 50μL of anhydrous ethanol and 2μL of sodium acetate, mix well, and place at -80℃ overnight. Centrifuge at 4℃, 12000rpm for 25 minutes, discard the supernatant; wash twice with 70% ethanol solution; leave in the air for 5 minutes, absorb excess alcohol, blow dry and add appropriate amount of enzyme-free water; measure concentration, detect by electrophoresis, and store at -80℃.

[0047] (2) Cas9 synthesis

[0048] 1) Plasmid linearization: Restriction endonuclease Xba I was purchased from NEB (NEB, Beijing). The linearization system is shown in Table 3.

[0049] Table 3 Linearization system

[0050]

[0051] Linearization conditions: 37°C water bath, 3h.

[0052] 2) Rubber cutting and recycling (rubber concentration: 1%);

[0053] 3) In vitro transcription;

[0054] The product recovered in the above step was used as a template for in vitro transcription. The in vitro mRNA transcription kit T7mMESSAGE The kit was purchased from Ambion (Ambion, USA), and the system is shown in Table 4.

[0055] Table 4 In vitro transcription system

[0056]

[0057] (3) Microinjection

[0058] Dilute gRNA and Cas 9 to 1000ng / μL. Mix 1μL of each at a 1:1 ratio and add about 0.4μL of phenol red solution (as an indicator). Transfer the mixture to a micro-needle tube and inject it into 1-cell to 4-cell fertilized eggs using a microinjector. At the same time, inject gRNA and Cas9 alone as a negative control. After the injection is completed, transfer it to a 28℃ constant temperature circulating water incubation system for incubation (after hatching).

[0059] 3. Mutation screening of F0 generation positive fish

[0060] (1) Design of primers for mutation detection

[0061] Primers were designed and tested for specificity using NCBI Primer blast (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ). PAGE primers are as follows:

[0062] pomc-PF: 5'-CTCTGTCACTCTGACCTCAC-3'; (SEQ ID NO.4)

[0063] pomc-PR: 5'-AGCGGAAGTGCTCCATTGAG-3'. (SEQ ID NO.5)

[0064] (2) F0 test:

[0065] On the third day after hatching, some fish eggs were collected and DNA was extracted using the phenol-chloroform-isoamyl alcohol extraction method. The injected fish eggs were collected and genomic DNA was extracted using the phenol-chloroform-isoamyl alcohol extraction method. This was used as a template and PCR amplification was performed using pre-designed restriction primers. The product was recovered and DNA was extracted. This was used as a template and primers were designed at both ends of the target site to perform PCR amplification of a 133bp size band. PAGE electrophoresis was performed after amplification. Due to sequence changes and the fact that the positive fish were chimeras, the mutant ribbon fish had a large number of new bands and heteroduplexes compared to the control.

[0066] 4. Cultivation of F2 tilapia with homozygous deletion mutation of pomc gene

[0067] (1) Obtain heterozygous pomc mutation F1 generation;

[0068] When the male fish reached sexual maturity (about 6 months old), they were mated with wild-type female fish to obtain F1 broodstock with different mutation types. The mutation types were identified by PAGE primer amplification combined with sequencing technology, and male and female tilapia with the same mutation type of frameshift mutation (base deletion not a multiple of 3) were selected as F1 broodstock.

[0069] (2) Obtaining homozygous pomc mutation F2 generation

[0070] When the F1 generation broodstock reaches sexual maturity, the two can mate to obtain the F2 generation fish containing homozygous deletions. Homozygous mutations are detected using PAGE combined with sequencing technology. First, the genomic DNA of the F2 generation fish is extracted, and it is amplified using PAGE primers as a template and the amplified product is subjected to PAGE, and then imaged by a gel imaging system. The wild type (one band), heterozygote (4 bands) and homozygote (1 band) are judged by the band display. The tail fin genome of the homozygous fish detected initially is extracted, amplified with PAGE primers, recovered, subcloned, and sent to a sequencing company (Qingke, China) for sequencing to confirm the test results.

[0071] like Figure 1 As shown, the gRNA target sequence constructed in the third exon of the pomc gene of the present invention ( Figure 1 A). Screen the F1 generation of male and female fish with the same mutation type and use them as parents to crossbreed to obtain the F2 generation ( Figure 1 (B). PAGE results showed that the wild type had only one band; the heterozygote contained four bands: two heteroduplexes at the top, a wild type band at the bottom, and a mutant band; the homozygote had only one mutant band ( Figure 1 C). Homozygotes were identified by Sanger sequencing ( Figure 1D). Comparison of sequencing peaks revealed a 14 bp deletion at the homozygous target site, and this mutation would cause pomc to form a non-functional truncated protein, indicating that the present invention successfully established a pomc homozygous mutant line in Nile tilapia.

[0072] Example 2 Growth experiment based on pomc homozygous mutant fish

[0073] The obtained F2 generation was mixed and fed three times a day (9:00, 13:00, and 19:00). At 40 dpf, the mutant type was screened and the pomc was preliminarily weighed and counted. - / - 、pomc + / - and WT tilapia weight and length, the results are shown in Figure 2 and Figure 3 , the specific measurement method is as follows:

[0074] Body length (cm): Place the fish on a table, set aside a ruler, and accurately measure the length from the tip of the fish's mouth to the base of the tail (body length accurate to 1 decimal place, unit: cm).

[0075] Body weight (g): Fast overnight before weighing, scoop out the fish one by one with a net and wipe off the moisture on the body surface with a towel, and use a small scale to weigh each individual accurately (weight accurate to two decimal places, unit: g).

[0076] Body width (cm): Measure the vertical distance from the 2nd to 3rd fin ray of the fish's dorsal fin to the belly of the fish (body width is accurate to 1 decimal place, unit: cm).

[0077] Body depth (mm): Place the fish upright and use an electronic vernier caliper to accurately measure the body thickness (body width accurate to 1 decimal place, unit: mm).

[0078] like Figure 2 As shown, at 40 dpf, 40 days after hatching and co-culture, pomc - / - Tilapia weight and length were significantly higher than those of wild type and pomc + / - Tilapia, and pomfret + / - There was no significant difference in body weight and length between the two groups.

[0079] Keep pomc - / - When the pomc homozygous mutant was co-cultured with WT tilapia until 90 dpf, the weight, length, width and thickness of the pomc homozygous mutant were significantly greater than those of the wild type ( Figure 3 ).

[0080] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for creating a new fast-growing tilapia strain, characterized in that: The following steps are involved: (1) The fragment of the third exon of the pomc gene as shown in SEQ ID NO.1 was used as the gRNA target sequence; (2) gRNA primer sequences as shown in SEQ ID NOs. 2 and 3 were designed and synthesized according to the gRNA target sequence; (3) using gRNA primer sequence and gRNA plasmid amplification and then transcription and purification to obtain gRNA fragments; (4) transferring the gRNA fragment into the fertilized eggs of tilapia by gene editing, and obtaining F0 generation positive fish by primer detection as shown in SEQ ID NOs. 4 and 5 after incubation; (5) The positive male fish of the F0 generation are hybridized with the wild-type female fish to select the F1 generation broodstock, and the F1 generation broodstock are raised to sexual maturity and then mated to obtain the F2 generation tilapia with the homozygous deletion mutation.

2. The method according to claim 1, characterized in that: The amplification reaction system in step (3) is as follows: 2×TaqMaster Mix 25 μL, gRNAF / R 1 μL, gRNA plasmid 0.3 μL, and finally filled to 50 μL with RNase-free water.

3. The method according to claim 1, characterized in that: The reaction conditions in step (3) are: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 15 s, and 38 cycles; 72°C for 10 min, and 12°C for 10 min.

4. The method according to claim 1, characterized in that: The transcription system in step (3) includes: 1 μL of T7 / SP6 RNA polymerase, 2 μL of 10×Transcription buffer, 2 μL of 10×rNTP Mix, X μL of RNase inhibitor, and (14-x) μL of RNase-free water.

5. The method according to claim 1, characterized in that In step (4), the Cas9 protein and gRNA are mixed and injected into tilapia fertilized eggs.

6. The method according to claim 5, characterized in that The concentrations of Cas9 protein and gRNA are both 800-1000 ng / μL, and the mixed volume ratio of the two is 1:

1.

7. The method according to claim 1, characterized in that The target fragment amplified by the primer in step (4) is 133 bp. When a new unknown band and a heteroduplex appear in the test object compared with the control group, it is judged to be a mutant F0 generation positive fish.

8. The method according to claim 1, characterized in that When screening F1 generation broodstock in step (5), male and female tilapia with frameshift mutations of the same mutation type are used as F1 generation broodstock.

9. Use of the pomc gene knockout tilapia mutant obtained by the method according to any one of claims 1 to 8 in constructing an animal model related to pomc gene knockout syndrome or in drug screening.

Images