Silkworm gene precise knockout system based on CRISPR/cas9 double nickase technology and its application

A nicking enzyme, phs-bcr-lw062-cas9 technology, applied in the field of molecular biology, can solve the problems of low specificity, CRSIPR/Cas9 off-target mutation, etc., and achieve the effect of simple reaction process, reduced off-target rate, and increased expression efficiency

Active Publication Date: 2022-07-01
JIANGSU UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] As the third-generation gene editing technology, CRISPR / Cas9 system is based on the combination of simple nucleotide complementary pairing to achieve site-directed mutagenesis at genomic target sites. It has the advantages of simple experimental process, short time-consuming and small workload, and has been successfully Applied to genome editing in a variety of organisms; however, CRSIPR / Cas9 itself has a tendency for off-target mutations and relatively low specificity

Method used

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  • Silkworm gene precise knockout system based on CRISPR/cas9 double nickase technology and its application
  • Silkworm gene precise knockout system based on CRISPR/cas9 double nickase technology and its application
  • Silkworm gene precise knockout system based on CRISPR/cas9 double nickase technology and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Vector construction and preparation of knockout system

[0025] Taking the silkworm BmBLOS2 gene as an example, the test steps are as follows:

[0026] 1 Design and selection of targeting gRNA

[0027] The BmBLOS2 gene sequence was found in the silkworm genome database, and the upstream and downstream regions of the first and second exons of the gene were selected to design target sites. The gRNA sequence was designed by the online design tool (http: / / crispr.dbcls.jp), and sticky ends were added to both ends of the gRNA sequence. The primer sequences were designed as follows:

[0028]

[0029] Reverse primer → NNNNNNNNNNNNNNNNNNNNCAAA

[0030] A site needs a pair of gRNAs, and the PAMs of each other can be combined head-to-tail, head-to-head or tail-to-tail, with an interval of about 4-20bp. The selected target site sequences are shown in Table 1:

[0031] Table 1 Targeting gRNA sequences

[0032]

[0033] 2 gRNA synthesis and vector construction

...

Embodiment 2

[0058] Example 2 Targeted knockout of the silkworm BmBLOS2 gene

[0059] The knockout system established in Example 1 was used to construct site-directed knockout vectors of the corresponding genes, respectively, and transfected silkworm ovary cells (BmN).

[0060] 1 BmN cell line pre-experiment

[0061] 1.1 Mycoplasma detection: Aspirate 1 mL of BmN cell culture medium supernatant, use mycoplasma-specific primers for PCR amplification, and use agarose gel electrophoresis to identify whether there is mycoplasma contamination in the cell line;

[0062] 1.2. Determination of growth rate: BmN cells were subcultured to observe the growth state and proliferation rate of BmN cells.

[0063] 2 Transfection

[0064] 2.1 Cell plating: Press 10 for cells before transfection 5 Cells / mL were plated until the cell confluence was 60-80% the next day.

[0065] 2.2 Dilute 0.5 μg of plasmid pHS-BCR-LW062 with 25 μL of Opti-MEM medium and mix thoroughly;

[0066] 2.3 Dilute 1.25 μL of Entr...

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Abstract

The invention discloses a silkworm gene precise knockout system based on CRISPR / Cas9 double nickase technology. The system includes a Cas9 mutant expression vector pHS-BCR-LW062-cas9 (full length 8654bp) and a sgRNA (guide sequence) production vector pHS ‑BPR‑LC001 (full length 4893bp). The silkworm gene precise knockout system based on CRISPR / Cas9 double nickase technology of the present invention can target and knock out silkworm functional genes, and greatly reduce the off-target rate, at least 50 times, or even 1500 times; the system is extremely The accuracy rate of gene knockout is greatly improved, and the reaction process is simple; the Cas9 mutant expression vector pHS-BCR-LW062-cas9 in the system of the present invention introduces the strong promoter IE1 of the silkworm nuclear polyhedrosis virus and silkworm codons The optimized cas9D10A greatly increases the expression efficiency of the enzyme; the pHS-BPR-LC001 in the system of the present invention uses the U6 promoter of Bombyx mori, and is inserted into the IIS type Bsa I enzyme cleavage site, and the golden gate ligation method can be used. The rapid construction of sgRNA expression vector greatly saves the operation steps.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and relates to a CRISPR / Cas9 double nickase technology, in particular to a silkworm gene precise knockout system based on the CRISPR / Cas9 double nickase technology and an application thereof. Background technique [0002] Genome editing technology is a genetic manipulation technology that modifies DNA sequences at the genome level, and can achieve precise manipulations such as gene site-directed insertion or deletion mutation, gene knockout, multi-site or multi-gene mutation and fragment deletion. [0003] As a third-generation gene editing technology, the CRISPR / Cas9 system is based on a simple nucleotide complementary pairing method to achieve site-directed mutagenesis at target sites in the genome. It has the advantages of simple experimental process, short time-consuming and low workload, and has been successfully used. Genome editing applied to a variety of organisms; however, CRSIP...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/90
CPCC12N15/85C12N15/902C07K14/43586C12N2810/10C12N2800/22C12N2800/105
Inventor 沈兴家陈艳荣朱娟蒋涛唐顺明赵巧玲
Owner JIANGSU UNIV OF SCI & TECH
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