Marker gene of CRISPR/Cas9 gene editing system applied to hermetia illucens

A gene editing and black soldier fly technology, applied in genetic engineering, hydrolytic enzymes, using microinjection methods, etc., can solve the problems of complex mutation screening, achieve excellent results, and facilitate gene function research

Inactive Publication Date: 2020-12-22
江苏绿博低碳科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] After extensive research and repeated experiments, the inventors found that the marker gene is an effective method to solve the complex problem of screening mutations in the above-mentioned black soldier fly gene editing process

Method used

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  • Marker gene of CRISPR/Cas9 gene editing system applied to hermetia illucens
  • Marker gene of CRISPR/Cas9 gene editing system applied to hermetia illucens
  • Marker gene of CRISPR/Cas9 gene editing system applied to hermetia illucens

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Experimental program
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Effect test

Embodiment 1

[0030] The acquisition of embodiment 1 primer

[0031] 1) Extract the total RNA of the black soldier fly in the late stage of embryonic development, reverse transcribe it into cDNA, and clone the white eye color gene white from the black soldier fly cDNA with primers white-F:ATGACTCCAGGCAGCGACGA and white-R:TTAGTTGGCGCCTCTCGCTT, and connect it to P-Jet- 1.2 Sequencing verification on the carrier, the obtained gene sequence is shown as SEQ NO.1 in the sequence listing.

[0032] 2) Select a target site with a length of 23 bases and a PAM sequence of NGG (5'-3') on a single exon of the white gene, and the selected two target site sequences are target site 1 (Target site1:3 'GGCGAACTGCTTGCCATCATGGG 5') and target site 2 (Target site2: 3'GGAGCACCAGGACGTATTAAAGG 5').

[0033] 3) Design primers to synthesize corresponding sgRNA templates, the primer sequences are:

[0034] sgRNA1-F, as shown in SEQ NO.2 in the sequence listing;

[0035] sgRNA2-F, as shown in SEQ NO.3 in the sequen...

Embodiment 2

[0038] Example 2 sgRNA template acquisition

[0039] The template primers sgRNA1-F and sgRNA2-F were respectively annealed and extended with the universal reverse primer to obtain the template (Toyobo).

[0040] The reaction system is as follows:

[0041]

[0042] The PCR reaction conditions are as follows: 94° C. for 2 min; 20 cycles of 94° C. for 15 s, 55° C. for 30 s, and 68° C. for 10 s; 68° C. for 5 min.

[0043] The recovered and purified PCR product is the template sgRNA.

[0044] The sequence of the template sgRNA is:

[0045] sgRNA-1: as shown in SEQ NO.5 in the sequence listing;

[0046] sgRNA-2: as shown in SEQ NO.6 in the sequence listing.

Embodiment 3

[0047] Example 3 A large amount of synthesis of sgRNA

[0048] The obtained sgRNA template was amplified in vitro through the kit to obtain sgRNA in a single-stranded state【 T7 Kit (Ambion)]. The reaction system is as follows:

[0049]

[0050] After mixing, react at 37°C for 12 hours and then purify to obtain sgRNA.

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Abstract

The invention discloses a method for screening CRISPR / Cas9 gene edited hermetia illucens. The method comprises the following steps: (1) respectively performing PCR on template primers sgRNA1-F and sgRNA2-F in which white genes are knocked out and universal reverse primers, and recovering and purifying a PCR product to obtain template sgRNA; (2) performing in-vitro mass amplification on the sgRNAtemplate to obtain sgRNA in a single-stranded state; (3) mixing the sgRNA in the single-stranded state with a cas9 protein, and performing microinjection into newly-produced fertilized eggs of the hermetia illucens; and (4) performing dark treatment on the injected eggs until the eggs are incubated, observing the eye color phenotype of the incubated hermetia illucens, and picking out the individuals with whitened eyes. According to a gene editing technology, eye color related genes are successfully knocked out, the obtained gene mutation hermetia illucens can be judged by observing whether whitened eyes appear or not, phenotype is easy to observe in the early stage, the gene mutation hermetia illucens has an excellent effect when being used as a marker gene, and convenience can be providedfor subsequent hermetia illucens gene function research.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a marker gene of a CRISPR / Cas9 gene editing system applied to a black soldier fly. Background technique [0002] The black soldier fly, whose scientific name is Hermetia illucens, belongs to Diptera and Soldieridae insects, and is an important resource insect. The larvae feed on decaying organic matter and animal waste in nature. Their habit has great application value in environmental governance. The black soldier fly has a life cycle of 40-45 days. It has strong reproductive ability and adaptability. It can be raised artificially on a large scale, and can efficiently process organic waste such as kitchen waste and livestock manure. In addition, black soldier fly larvae can significantly reduce the pathogenic bacteria in the feces and inhibit the reproduction of houseflies during the process of feeding on livestock and poultry manure. [0003] Black soldier fly larvae...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N9/22C12N15/89C12N15/113C12N15/11A01K67/027
CPCC12N15/902C12N9/22C12N15/89C12N15/113A01K67/0339C12N2310/10A01K2217/07A01K2227/706A01K2267/02
Inventor 黄勇平詹帅王四宝寇宗庆许军
Owner 江苏绿博低碳科技股份有限公司
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