A method for targeted creation of new strains of fast-growing rohu (labeo rohita)

Abstract

The application discloses a method for creating a new strain of fast-growing Nile tilapia, and belongs to the technical field of fish breeding. The method comprises the following steps: (1) taking a fragment as shown in SEQ ID NO. 1 in the third exon of the pomc gene as a gRNA target sequence; (2) designing and synthesizing a gRNA primer sequence; (3) obtaining a gRNA fragment by transcription and purification after amplification of the gRNA primer sequence and a gRNA plasmid; (4) transferring the gRNA fragment into fertilized eggs of the Nile tilapia through gene editing to obtain F0 generation positive fish; and (5) crossing the F0 generation positive male fish with wild-type female fish, screening F1 generation parent fish, and cultivating the F1 generation parent fish to obtain F2 generation Nile tilapia with homozygous deletion mutation. The application establishes a pomc homozygous knockout strain in the Nile tilapia for the first time, and the new fast-growing strain is a complete gene function loss model, has lower cost and higher efficiency, and the phenotype can be stably inherited.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fish cultivation, and particularly relates to a method for creating a new strain of fast-growing Oreochromis niloticus. BACKGROUND

[0002] Excellent traits of fish are closely related to the economic benefits of aquaculture. In order to further improve the yield of aquatic products in China, great progress has been made in breeding technology research and development and excellent strain cultivation in recent years.

[0003] Hybridization and selection are the main ways of fish breeding. However, the breeding cycle of hybridization is often long, and due to the existence of inter-specific reproductive isolation, it is difficult to form a fertile strain through distant hybridization. Therefore, the breeding method of hybridization 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 has no effect on fertility, which is conducive to the stable inheritance of new phenotypes.

[0004] Among the arcuate nucleus region, there are two types of neurons that are particularly important for controlling appetite, namely proopiomelanocortin (POMC) neurons and agouti-related peptide (AgRP) neurons. POMC neurons promote satiety, while AgRP neurons promote hunger. POMC neurons produce POMC protein, which is the precursor of alpha-melanocyte-stimulating hormone (alpha-MSH), endorphins, and adrenocorticotropic hormone (ACTH). Among them, alpha-MSH is an important activating ligand of melanocortin receptor-4 (MC4R) and plays a core role in body weight regulation (Erica J P Anderson et al. Regulation of feeding and energy homeostasis by alpha-MSH. J Mol Endocrinol. 2016 May; 56(4): T157-74. doi: 10.1530 / JME-16-0014). Injection of synthetic alpha-MSH antagonists 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] Previous studies have shown that POMC mutants were constructed by targeting plasmids in mice, and it was found that deletion of POMC in mice leads 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 cause 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 Mori 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 there is no mature conditional knockout technology in other species such as fish. In addition, after injecting pomc siRNA into the ventricle of the mandarin fish, the feeding amount and domestication rate of the mandarin fish can be improved (Lu Haolin. The effect of pro-opiomelanocortin on the domestication of mandarin fish and its regulatory pathways. Central China Agricultural University. June 2021). The above two methods can simply and conveniently silence the function of POMC, but still have the problems of limited efficiency and cannot be stably inherited. Therefore, how to stably and efficiently knockout the pomc gene in fish still needs further exploration. SUMMARY

[0006] In view of the above problems in the prior art, the present application provides a method for targeted creation of fast-growing new strains of tilapia, which can efficiently and stably knockout the pomc gene without affecting other genes.

[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application to solve its technical problems is:

[0008] The purpose of the present application is to provide a method for targeted creation of fast-growing new strains of tilapia, comprising the following steps:

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

[0010] (2) The gRNA primer sequences as shown in SEQ ID NO. 2 and 3 were designed and synthesized according to the gRNA target sequence;

[0011] (3) The gRNA fragment was obtained by transcription and purification after amplification of the gRNA primer sequence and the gRNA plasmid;

[0012] (4) The gRNA fragment was introduced into the fertilized eggs of the tilapia through gene editing, and the F0 generation positive fish was obtained by detecting the primers as shown in SEQ ID NO. 4 and 5 after incubation;

[0013] (5) The F0 generation positive male fish was crossed with the wild type female fish, and the F1 generation parent fish was screened out, which was bred to sexual maturity and then mated to screen out the F2 generation tilapia with homozygous deletion mutation.

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

[0015] Further, the reaction conditions in step (3) are as follows: 95℃ pre-denaturation for 3min; 95℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 15s, cycle 38 times; 72℃ for 10min, 12℃ for 10min.

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

[0017] Further, the Cas9 protein and the gRNA were mixed and injected into the fertilized eggs of the tilapia in step (4).

[0018] Further, the concentration of the Cas9 protein and the gRNA is 800-1000 ng / μL, and the volume ratio of the mixture of the two is 1:1.

[0019] Further, the amplification target fragment of the primer in step (4) is 133bp, and when a large number of new unknown bands and heteroduplexes appear in the detection object compared with the control group, it is judged to be the F0 generation positive fish with mutation.

[0020] Further, in step (5), the F1 generation brood fish are screened, and the male and female tilapia with the same type of shift mutation are used as the F1 generation brood fish.

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

[0022] The present application has the following advantages:

[0023] The present application mutates the pomc gene of tilapia through CRISPR / Cas technology, and it is found that the pomc knockout significantly improves the growth rate at 40 days and 90 days after hatching. Compared with the previous gene knockout technology and knockdown, the present application has the characteristics of low cost, small toxicity, high accuracy, high efficiency, easy operation, etc., and the obtained phenotype can be stably inherited, which is beneficial to the cultivation of new strains.

[0024] The present application designs a unique sgRNA sequence on the pomc gene, which can quickly and efficiently knockout the pomc gene in tilapia without affecting other genes, forming a pomc gene specific knockout tilapia.

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

[0026] Figure 1 For the establishment of a pomc homozygous knockout line in Nile tilapia; wherein, A) pomc gene structure and mutation detection; the target site is on the third exon, and the Sanger sequencing result shows different mutation types, the PAM region is marked with red font, and the deleted bases are marked with (-); B) construction of pomc homozygous mutant line, wild type, heterozygous mutant and homozygous mutant 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 wild type (WT) tilapia growth experiment box plot; wherein, 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 90 dpf pomc - / - and wild type (WT) tilapia growth experiment box plot; wherein, 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 embodiments of the present application are described below to facilitate the understanding of the present application for those skilled in the art, but it should be clear that the present application is not limited to the scope of the specific embodiments, and for those skilled in the art, it is obvious that various changes are within the spirit and scope of the present application defined and determined by the appended claims, and all the inventions utilizing the concept of the present application are within the scope of protection.

[0030] Example one effective targeting of pomc gene of nile tilapia using CRISPR / Cas9 gene editing technology

[0031] 1. Target design

[0032] The full sequence of pomc gene of tilapia was downloaded on NCBI, and the target was designed on the third exon of pomc according to the CRISPR / gRNA target sequence design website (http: / / zifit.partners.org / ZiFiT / ), the target sequence was: 5'-CCAGCTCTGTCACTCTGACCTCAC-3'(SEQ ID NO.1), and the gRNA primer sequence was synthesized after design.

[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, the designed F primer and universal R primer were used for amplification and the fragment was recovered. Taq DNA polymerase (2 x 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 °C for 3 min; 95 °C for 30 s, 60 °C for 30 s, 72 °C for 15 s, cycle 38 times; 72 °C for 10 min, 12 °C for 10 min.

[0042] 2) In vitro transcription

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

[0044] Table 2 In vitro transcription reaction system

[0045]

[0046] After mixing, 37 °C water bath for 2 h. Add 2 μL of DNase I, 37 °C water bath for 15 min. Add 50 μL of anhydrous ethanol and 2 μL of sodium acetate, mix well, -80 °C overnight. 4 °C, 12000 rpm centrifugation for 25 min, discard the supernatant; volume concentration 70% ethanol solution wash 2 times; empty for 5 min, absorb the excess alcohol, super clean dry and add appropriate amount of enzyme-free water; measure the concentration, electrophoresis detection, -80 °C preservation.

[0047] (2) Cas9 synthesis

[0048] 1) Plasmid linearization: restriction enzyme Xba I was purchased from NEB (NEB, Beijing), and the linearization system is shown in Table 3.

[0049] Linearization system

[0050]

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

[0052] 2) Cut and recover the gel (gel concentration: 1%);

[0053] 3) In vitro transcription;

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

[0055] Table 4 In vitro transcription system

[0056]

[0057] (3) Microinjection

[0058] The gRNA and Cas 9 were diluted to 1000 ng / μL. According to the ratio of 1:1, 1 μL each was mixed, and about 0.4 μL of phenol red solution (as an indicator) was added. The mixture was transferred to a microsyringe and injected into 1-4 cell zygotes using a microinjection instrument, and gRNA and Cas9 were injected alone as a negative control. After injection, it was transferred to a 28°C constant temperature circulating water incubation system for incubation (incubated to post membrane).

[0059] 3. Mutation screening of F0 positive fish

[0060] (1) Mutation detection primer design

[0061] The primer was designed on NCBIPrimer blast (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) and its specificity was detected. The PAGE primer is 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 detection:

[0065] On the third day after hatching, some fish eggs were collected, and DNA was extracted using the phenol-chloroform-isoamyl alcohol extraction method. Genomic DNA was extracted from the injected fish eggs using the same method. Using this DNA as a template, PCR amplification was performed with pre-designed enzyme digestion primers, and the product was recovered. DNA was extracted, and primers were designed at both ends of the target site to amplify a 133bp band using PCR. After amplification, PAGE electrophoresis was performed. Due to the sequence change and the fact that the positive fish were chimeras, the mutant banded fish showed a large number of new bands and heteroduplexes compared to the control fish.

[0066] 4. Breeding F2 generation tilapia with homozygous deletion mutation of the pomc gene

[0067] (1) Obtain the F1 generation of heterozygous pomc mutants;

[0068] Once the male tilapia reach sexual maturity (around 6 months old), they are mated with wild-type female tilapia to obtain F1 generation parent fish with different mutation types. The mutation types are identified by PAGE primer amplification combined with sequencing technology. Male and female tilapia with the same frameshift mutation (non-3 fold deletion of base) are selected as F1 generation parent fish.

[0069] (2) Obtain homozygous pomc mutant F2 generation

[0070] Once the F1 generation parent fish reach sexual maturity, mating them yields the F2 generation fish containing homozygous deletions. Homozygous mutations are detected using PAGE combined with sequencing technology. First, genomic DNA is extracted from the F2 generation fish. Using this DNA as a template, PAGE primers are applied to amplify the DNA, and the amplified products are then PAGE-enhanced and imaged using a gel imaging system. Banding is used to identify wild-type (one band), heterozygous (four bands), and homozygous (one band). For the preliminarily identified homozygous fish, the caudal fin genome is extracted, amplified using PAGE primers, recovered, subcloned, and sent to a sequencing company (Qingke, China) for sequencing to confirm the results.

[0071] like Figure 1 As shown, the gRNA target sequence constructed in the third exon of the pomc gene in this invention ( Figure 1 (A). Select F1 generation male and female fish with the same mutation type and use them as parents to crossbreed to obtain F2 generation ( Figure 1 PAGE results showed that the wild type had only one band; the heterozygote had four bands: two heteroduplexes at the top, one wild-type band and one mutant band at the bottom; the homozygote had only one mutant band. Figure 1 (C). Homozygotes were identified using Sanger sequencing. Figure 1Figure 6. Sequencing peak plot of pomc homozygous mutant. The sequencing peak plot comparison found a 14 bp deletion at the homozygous target site, which would result in a truncated protein of pomc without function. This indicated that the pomc homozygous mutant line was successfully established in Nile tilapia.

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

[0073] The obtained F2 generation was mixedly raised and fed three times a day (9:00; 13:00; 19:00). At 40 dpf, the mutation types were screened and the pomc - / - , pomc + / - and WT tilapia were weighed and measured for body length. Figure 2 and Figure 3 , and the specific measurement methods are as follows:

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

[0075] Body weight (g): The fish were fasted overnight before weighing, and then the fish were caught one by one with a net and the body surface water was wiped off with a towel. A small body weight scale was used to accurately weigh each individual (body weight accurate to two decimal places, unit: g).

[0076] Body width (cm): Measure the vertical distance from the head number 2-3 fin bar of the dorsal fin to the abdomen of the fish (body width accurate to 1 decimal place, unit: cm).

[0077] Body depth (mm): The fish was placed vertically, and the body depth was accurately measured using an electronic vernier caliper (body width accurate to 1 decimal place, unit: mm).

[0078] As shown in Figure 2 , at 40 dpf, after 40 days of mixed rearing after hatching, it was found that the pomc - / - tilapia had significantly higher body weight and body length than the wild type and pomc + / - tilapia, while there was no significant difference in body weight and body length between pomc + / - and WT.

[0079] The pomc - / - and WT tilapia were continued to be mixedly raised to 90 dpf, and it was found that the pomc homozygous mutant had significantly higher body weight, body length, body width and body depth than the wild type Figure 3 .

[0080] Finally, it should be noted that the above detailed description is merely illustrative of the technical solutions of the present application and is not limiting, and although the present application has been described in detail with reference to the examples, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present application, and all should be covered in the scope of the claims of the present application.

Claims

1. A method of targeted creation of a new strain of fast-growing Nile tilapia, characterized in that, Comprising the following steps: (1) with Pomc the fragment as shown in SEQ ID NO. 1 on the third exon of the gene as the gRNA target sequence; (2) The gRNA primer sequences shown as SEQ ID NO. 2 and 3 were designed and synthesized according to the gRNA target sequence; (3) The gRNA fragment was obtained by transcription purification after amplification of the gRNA primer sequence and gRNA plasmid; (4) The gRNA fragment was introduced into the fertilized eggs of tilapia by gene editing, and after incubation at 28℃ in a constant temperature circulating water, the F0 generation positive fish was detected by primers shown as SEQ ID NO. 4 and 5; (5) The F0 generation positive male fish was crossed with wild type female fish, and the F1 generation parent fish was screened out, which was bred to sexual maturity and then mated to screen out F2 generation tilapia with homozygous deletion mutation.

2. The method of claim 1, wherein, The amplification reaction system in step (3) is as follows: 2x TaqMaster Mix 25 μL, gRNA F / R 1 μL, gRNA plasmid 0.3 μL, and finally supplemented with RNase-free water to 50 μL.

3. The method of claim 1, wherein, The reaction conditions in step (3) are as follows: 95 ℃ pre-denaturation for 3 min; 95 ℃ denaturation for 30 s, 60 ℃ annealing for 30 s, 72 ℃ extension for 15 s, cycle 38 times; 72 ℃ for 10 min, 12 ℃ for 10 min.

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

5. The method of claim 1, wherein, In step (4), the Cas9 protein and gRNA were mixed and injected into the fertilized eggs of tilapia.

6. The method of claim 5, wherein, The concentration of Cas9 protein and gRNA is 800-1000 ng / μL, and the volume ratio of the mixture is 1:

1.

7. The method of claim 1, wherein, In step (4), the amplification target fragment of the primer is 133 bp, and when a new unknown band and heteroduplex appear compared with the control group, it is judged to be the F0 generation positive fish with mutation.

8. The method of claim 1, wherein, In step (5), the same type of frameshift mutation male and female tilapia were used as F1 generation parent fish when screening F1 generation parent fish.

9. The method according to any one of claims 1 to 8, wherein the method is performed in vitro. Pomc The use of the gene knockout tilapia mutants in constructing animal models related to Pomc gene knockout syndromes or in drug screening.

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