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Application of CRISPR (clustered regularly interspaced short palindromic repeats)/Cpf1 system with compounded crRNA in gene editing

A technique of cpf1 and chemical synthesis, applied in the application of CRISPR/Cpf1 system in gene editing, in the field of chemically synthesized crRNA, which can solve problems such as inability to cut

Active Publication Date: 2016-08-31
SUZHOU GENEPHARMA
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  • Summary
  • 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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  • Application of CRISPR (clustered regularly interspaced short palindromic repeats)/Cpf1 system with compounded crRNA in gene editing
  • Application of CRISPR (clustered regularly interspaced short palindromic repeats)/Cpf1 system with compounded crRNA in gene editing
  • Application of CRISPR (clustered regularly interspaced short palindromic repeats)/Cpf1 system with compounded crRNA in gene editing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1. Detecting the gene editing ability of AsCRISPR / Cpf1 system, FnCRISPR / Cpf1 system, LbCRISPR / Cpf1 system and Lb2CRISPR / Cpf1 system

[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 capabilities of the AsCRISPR / Cpf1 system, FnCRISPR / Cpf1 system, LbCRISPR / Cpf1 system and Lb2CRISPR / Cpf1 system. The AsCRISPR / Cpf1 system is from Acidaminococcus_sp.BV3L6, which expresses the AsCpf1 protein shown in sequence 6 in the sequence listing; the FnCRISPR / Cpf1 system comes from Francisella_novicida, which expresses the FnCpf1 protein shown in sequence 5 in the sequence listing; the LbCRISPR / Cpf1 system comes from Lachnospiraceaebacterium ND2006 , which expresses the LbCpf1 protein shown in sequence 3 in the sequence listing; the Lb2CRISPR / Cpf1 system is from Lachnospiraceae_bacterium_MA2020, which expresses the Lb2Cpf1 protein shown in sequence 4 in the sequence listing.

[0090] T...

Embodiment 2

[0223] Example 2. Application of chemically synthesized crRNA for CRISPR / Cpf1 system in gene editing

[0224] 1. Preparation of chemically synthesized crRNA

[0225] Synthesize the crRNAs shown in Table 1. In Table 1, the single underline is the 2nd to 21st position from the 5' end of the Lb crRNA backbone sequence, the dotted line is the 2nd to 20th position from the 5' end of the Lb2crRNA backbone sequence, and the double underline is the target sequence I from the 5' end Positions 1 to 19, the box is the 1st to 23rd position from the 5' end of the target sequence I, the single wavy line is the 1st to 23rd position from the 5' end of the target sequence II, and the double wavy line is the 5' end of the target sequence II Numbers 1 to 19 from the end.

[0226] All the crRNAs shown in Table 1 are in one tube of 1OD (33 μg), and 66 μL of DEPC water is added to each tube to obtain an RNA solution with an RNA concentration of 0.5 μg / μL.

[0227] Table 1. Basic information of c...

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Abstract

The invention discloses application of a CRISPR (clustered regularly interspaced short palindromic repeats) / Cpf1 system in gene editing. The CRISPR / Cpf1 system comprises a1), chemosynthetic crRNA or a2), a crRNA expressing carrier. The CRISPR / Cpf1 system can be c1), an LbCRISPR / Cpf1 system, c2), an Lb2CRISPR / Cpf1 system, c3), an FnCRISPR / Cpf1 system or c4), an AsCRISPR / Cpf1 system. Experiments prove that the chemosynthetic crRNA can play an effective role in guidance of Cpf1 cut-editing specific target sites, and efficiency is high. Applying the crRNA expressed by the carrier or the chemosynthetic crRNA to the CRISPR / Cpf1 system has high application value in gene editing.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of chemically synthesized crRNA to CRISPR / Cpf1 system in gene editing. 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 effe...

Claims

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Application Information

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IPC IPC(8): C12N15/85
CPCC12N15/85C12N2810/80
Inventor 张佩琢王德华徐明亮
Owner SUZHOU GENEPHARMA
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