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Applications of modified crRNA in CRISPR/Cpf1 gene editing system

A cpf1, gene editing technology, applied in DNA/RNA fragments, genetic engineering, recombinant DNA technology, etc., can solve problems such as inability to cut

Inactive Publication Date: 2016-12-21
SUZHOU GENEPHARMA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the CRISPR / Cas9 system also has some shortcomings, such as the Cas9 protein cannot cut arbitrary sequences, and the 3' end of the target must contain a PAM sequence (such as the SpCas9 protein requires the PAM sequence to be NGG)

Method used

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  • Applications of modified crRNA in CRISPR/Cpf1 gene editing system
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  • Applications of modified crRNA in CRISPR/Cpf1 gene editing system

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Embodiment 1

[0088] Example 1. Application of chemically synthesized and modified crRNA for CRISPR / Cpf1 system in gene editing

[0089] The hAAVS1 gene (Gene ID: 54776) and THUMPD3-AS1 gene (Gene ID: 440944) were selected as the target genes for detecting the gene editing ability of the CRISPR / Cpf1 system. The CRISPR / Cpf1 system is specifically AsCRISPR / Cpf1 system or FnCRISPR / Cpf1 system. The AsCRISPR / Cpf1 system is derived from Acidaminococcus_sp.BV3L6, which expresses the AsCpf1 protein shown in Sequence 2 in the Sequence Listing. The FnCRISPR / Cpf1 system is from Francisella_novicida, which expresses the FnCpf1 protein shown in sequence 3 in the sequence listing.

[0090] The target sequence I was selected according to the nucleotide sequence of the hAAVS1 gene, and the target sequence II was selected according to the nucleotide sequence of the THUMPD3-AS1 gene. The sequences of target sequence I and target sequence II are as follows:

[0091] Target sequence Ⅰ: 5'-TCTGTCCCCTCCACCCAC...

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Abstract

The invention discloses applications of a CRISPR / Cpf1 system in gene editing. CrRNA in the CRISPR / Cpf1 system is modified crRNA; the modification manner is desoxyribonucleic acid modification; the desoxyribonucleic acid modification method comprises the step of increasing 1-3 desoxyribonucleic acid at the 5' terminal and / or increasing 1-3 desoxyribonucleic acid at the 3' terminal of crRNA; and the desoxyribonucleic acid is deoxythymidine acid. The experiment proves that the crRNA formed through chemical synthesis and having the modification can cause the mutation of an hAAVS1 gene and a THUMPD3-AS1 gene when used for the AsCRISPR / Cpf1 system or the FnCRISPR / Cpf1 system, namely, the modified crRNA has certain gene editing capacity when used for the AsCRISPR / Cpf1 system or the FnCRISPR / Cpf1 system, and therefore, the modified crRNA has a significant application value in gene editing utilizing the CRISPR / Cpf1 system.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of modified crRNA in a CRISPR / Cpf1 gene editing system. Background technique [0002] Gene editing is a technology for precise modification at the genome level, which can complete gene-directed deletion (InDel) mutation, gene-directed insertion mutation, simultaneous mutation of multiple sites, and deletion of small fragments. Gene editing technology can be used in the study of gene function and disease pathogenesis, the construction of disease animal models, biological therapy, research on genetic and tumor-related diseases, research on integrated viral diseases, and improvement of agricultural and livestock species. Gene editing technology is a tool for fundamentally changing the genetic material DNA of a species, and has extremely wide application value and development prospects. [0003] Zinc finger nuclease (ZFN), transcription activator-like effector nuclease (...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85
CPCC12N15/113C12N15/85C12N2310/10C12N2800/80C12N2810/10
Inventor 王德华徐明亮张佩琢
Owner SUZHOU GENEPHARMA
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