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CRISPR/Cas12a gene editing system and application thereof

A gene editing and gene technology, applied in the field of gene editing systems, can solve the problems of long cycle, high cost and low recombination frequency

Inactive Publication Date: 2019-04-23
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the low recombination frequency and high cost of the traditional homologous recombination system, long cycle and heavy workload in the operation process, and the off-target effect of Cas9, the recognition limitation of PAM, and not being suitable for point mutations, the present invention provides CRISPR / The Cas12a system has been optimized, and an efficient and specific genetic manipulation method has been invented and applied to genome editing of mammalian cells and Escherichia coli, providing a powerful tool for gene function research and metabolic engineering

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  • CRISPR/Cas12a gene editing system and application thereof

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Embodiment 1

[0036] 40 Cas12a protein sequences were collected from Uniprot, and a phylogenetic tree was constructed for analysis, among which LiCas12a was proved to have double-stranded DNA cutting activity. The specific method is as follows:

[0037] To find new functional Cas12a for genome editing, we collected 40 Cas12a protein sequences from Uniprot (https: / / www.uniprot.org) and analyzed these sequences together with AsCas12a, FnCas12a and LbCas12a by phylogenetic tree ( figure 1 ). Considering the different evolutionary origins and lengths of Cas12a proteins, five Cas12a proteins from Arcobacter butzleri L348, Eubacterium eligens, Francisella philomiragia subsp. philomiragia, Helcococcus kunzii and Leptospira inadaiserovar Lyme were selected as candidates. Among these five candidates, LiCas12a demonstrated DNA cleavage activity.

[0038] First clone LiCas12a into pGEX-6p-1 vector. The protein LiCas12a was expressed and purified using Ni affinity chromatography from E. coli BL21....

Embodiment 2

[0041] In order to verify the activity of LiCas12a in mammalian cells, that is, whether it has the function of cutting DNA, we verified it through the following experiments.

[0042] First, the screened LiCas12a gene (see SEQ ID No: 1 in the sequence listing) was cloned into the plasmid pLiCas12a with crRNA and N23 to select the DNMT1 gene as the knockout target (see the sequence listing in SEQ ID No: 4). The constructed LiCas12a plasmid targeting DNMT1 was transfected into 293T cells, and then the GFP-positive cells were sorted out for monoclonal culture. The genomic DNA of the monoclonal cells was extracted and used as a template to amplify DNMT1, and sequenced for verification.

[0043] The DNMT1 gene was successfully disrupted in 9 out of 10 monoclonal cell lines (90%). image 3 Representative sequencing results are shown. In Western blot analysis, DNMT1 expression was completely disrupted in DNMT1-deficient cells (Mut) compared with wild-type cells (WT) ( Figure 4 ). ...

Embodiment 3

[0045] CRISPR-LiCas12a system-mediated point and back mutations in mammalian cells.

[0046] TERT, encoding telomerase reverse transcriptase, is a component of telomerase, a ribonucleoprotein polymerase that maintains the ends of telomeres by adding the telomeric repeat TTAGGG21. The TERT promoter is frequently altered in many tumor types such as melanoma, hepatocellular carcinoma, glioma, urothelial carcinoma and renal cell carcinoma. The mutations of chr5, 1,295,228C>T and 1,295,250 C>T in the TERT promoter were the most important mutations. We tested the genomic status of TERT in bladder and hepatocellular carcinoma cell lines, selecting T24 and Huh7 as editing targets.

[0047] Two independent crRNAs were designed for the promoter region of the TERT gene (for the sequence, see SEQ ID No: 5 in the sequence listing). The bladder cancer cell line T24 (chr 5:1295228T) and the hepatocellular carcinoma cell line Huh7 (chr 5:1295228C) were selected as the editing objects. The ...

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Abstract

The invention relates to a CRISPR / Cas12a gene editing system and application thereof. The invention comprehensively utilizes biochemical, molecular biology, cell biology and other methods to screen and identify novel LiCas12a protein with endonuclease activity from a plurality of different bacteria. Based on LiCas12a, a CRISPR / LiCas12a gene editing system with high editing efficiency and high specificity is established in eukaryotic cells and prokaryotic cells separately, and genetic manipulation such as knockout, insertion and point mutation of genes is achieved; a molecular mechanism that the mutation of a TERT gene promoter promotes the cell proliferation of bladder cancer and liver cancer is disclosed. The genetic editing system based on CRISPR-LiCas12a provides a more accurate and efficient gene editing method for research fields such as disease pathogenesis and metabolic engineering transformation.

Description

technical field [0001] The present invention relates to a gene editing system, in particular to a brand-new highly specific CRISPR / Cas12a system. Background technique [0002] The first generation of gene editing technology is homologous recombination to establish animal gene knockout and gene knockin gene mutation models. Gene knockout and gene knockin are accomplished by DNA homologous recombination technology. This is a very complicated technique, which is generally used to establish animal models of genetic diseases, and it is difficult to be used clinically or in agriculture and animal husbandry. The second generation gene editing technology is zinc finger nuclease (zinc finger endonuclease, ZFN) and transcription activator-like effector nuclease (transcription activator-like effector nuclease, TALEN) technology. The principles of these two technologies are gene editing systems established by combining DNA nucleic acid binding proteins and endonucleases. Because thes...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N15/85C12N15/90
CPCC12N9/16C12N15/85C12N15/902C12N15/907C12N2800/80C12N2810/10
Inventor 喻长远杨昭阎秋韵沈宗毅张富涵张琨钟博白素杭魏振华
Owner BEIJING UNIV OF CHEM TECH
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