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Novel DNA (deoxyribonucleic acid) nucleic acid cleaving enzyme and application thereof

A polynucleotide and nucleic acid construct technology, applied in the field of new DNA nuclease cutting enzymes, can solve the problems of inability to recognize target genes and low editing efficiency

Active Publication Date: 2020-06-19
SHANDONG SHUNFENG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the expression of these two variants in plant cells is not the same as that in human cells. It has been reported in the literature that xCas9 has relatively low editing efficiency on target genes in plant cells, and it cannot recognize targets containing NG sequences in rice. Gene

Method used

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  • Novel DNA (deoxyribonucleic acid) nucleic acid cleaving enzyme and application thereof
  • Novel DNA (deoxyribonucleic acid) nucleic acid cleaving enzyme and application thereof
  • Novel DNA (deoxyribonucleic acid) nucleic acid cleaving enzyme and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0264] Example 1 Editing experiments in tomato of expression vectors containing different Cas9 variants

[0265] 1. Experimental materials

[0266] Tomato (Solanum lycopersicum cultivar Ailsa Craig), vector pCAMBIA1300, restriction enzymes EcoRI and HindIII, T4 ligase

[0267] 2. Carrier construction

[0268] Use the PCR method to mutate the corresponding mutation site, and connect it to the corresponding vector using double enzyme digestion and T4 ligase. For the structure of the editing tool, see figure 1 a.

[0269] 3. Experimental method

[0270] Two target genes SlBRI1 (regulating plant type) and SlRIN (regulating fruit ripening) in tomato were selected, and 11 sgRNAs were designed for these two genes, including NGG PAMs (AGG, TGG and GGG) and NG PAMs (TGC, TGA , GGT, AGA, GGC, AGT) (see Table 1), for each sgRNA, 40T1 transgenic tomato plants were generated respectively.

[0271] Table 1 Primers used to generate sgRNA plasmids and sequence in tomato

[0272]

[0...

Embodiment 2

[0288] Example 2 Editing experiment of expression vectors containing different Cas9 variants and ABE in tomato

[0289] 1. Plant material

[0290] Wild-type tomato (variety, Ailsa Craig), all transfected tomatoes were grown in a standard greenhouse (12 hours light at 26°C / 12 hours light at 20°C)

[0291] 2. Carrier Construction

[0292] PCR was used to complete the cloning of the coding region of ABE 7.10 (TadA-TadA7.10), and PCR was used to complete gene fusion and gene mutation. The promoters used were RPS5A, Pro35S, and AtU6. The primers are shown in Table 2, and the structure of the editing tool is shown in figure 1 b.

[0293] 3. Tomato transformation

[0294] The binary plasmid expressing sgRNA and ABE was transfected into Agrobacterium agrobacterium EHA105, and the tomato cotyledons were infected with the Agrobacterium.

[0295] 4. Mutation detection

[0296] DNA from tomato leaves was extracted by the CTAB method, and 14 gene regions containing sgRNA target sites...

Embodiment 3

[0300] Example 3 Editing experiment of expression vectors containing different Cas9 variants in Arabidopsis

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Abstract

The invention relates to a novel DNA (deoxyribonucleic acid) nucleic acid cleaving enzyme and application thereof, and particularly relates to a mutant gene editing protein and a fusion protein containing the mutant gene editing protein. The mutant gene editing protein and the fusion protein can identify more types of PAM (polyacrylamide) sequences, so that the gene editing range is expanded, theediting efficiency can be improved, and mutation of a target site can be accurately mediated. The mutant gene editing protein and the fusion protein can be widely applied to animal and plant cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a novel DNA nuclease cutting enzyme and its application. Background technique [0002] CRISPR / Cas is an immune mechanism from bacteria to degrade invading viral DNA or other foreign DNA. Apply this mechanism to animal and plant cells to cut target DNA. After the DNA is damaged, the cell will start its own repair mechanism, such as homologous recombination (HR), non-homologous end joining (NHEJ) and other repair mechanisms. In the repair process, errors such as base substitution, deletion or insertion will occur, so as to realize the mutation of gene function. At present, CRISPR-Cas9 gene editing technology is a technology for specific DNA modification of targeted genes, and this technology is also a cutting-edge method currently used in gene editing. [0003] The CRISPR-Cas9 system requires two steps to function: 1. Base-pairing binding between sgRNA and target gene; 2. Cas9 recogn...

Claims

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Application Information

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IPC IPC(8): C12N9/22C07K19/00C12N15/62C12N15/55C12N15/82A01H5/00A01H6/20A01H6/82
CPCC12N9/22C12N15/8213C07K2319/00
Inventor 不公告发明人
Owner SHANDONG SHUNFENG BIOTECH CO LTD
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