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A method for high-efficiency blood cell reprogramming and simultaneous gene editing

A gene editing and blood cell technology, applied in the field of cell reprogramming and gene editing, can solve the problem of cell acquisition traumatic process

Active Publication Date: 2021-12-24
INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are many problems with the use of fibroblasts. Fibroblasts are exposed to continuous UV radiation to accumulate more mutations, and cell harvesting is also a traumatic process.

Method used

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  • A method for high-efficiency blood cell reprogramming and simultaneous gene editing
  • A method for high-efficiency blood cell reprogramming and simultaneous gene editing
  • A method for high-efficiency blood cell reprogramming and simultaneous gene editing

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Effect test

Embodiment 1

[0061] Using the optimized vector combination, blood cell reprogramming and high-efficiency PRDM14 locus gene editing can be realized. For the PRDM14 gene locus, we designed sgRNA at the position 4bp before the stop codon TAG to generate DSB. Double-cut pDonor includes LHA sequence, an E2A-GFP-Wpre-polyA sequence and RHA sequence. If the cell has undergone gene editing, the sequence after gene editing at the PRDM14 site is PRDM14-E2A-GFP-Wpre-polyA( figure 1 ), in iPSCs, the expression of PRDM14 also causes the expression of GFP at the same time, and the efficiency of gene editing can be determined by detecting the proportion of GFP fluorescence positive cells by flow cytometry. We compared 3 vector combinations, including combination 1: OS 2ug+M 1ug+K 1ug+B 0.5ug+Cas9 3ug+sgRNA 1ug+pDonor 2ug; combination 2: OS 2ug+M 1ug+B 0.5ug+Cas9- K 4ug+sgRNA1ug+pDonor 2ug; combination 3: OS 2ug+M1ug+B 0.5ug+Cas9-K 4ug+sgRNA 1ug+pDonor 2ug+SV40LT 1ug. By counting clones 2 weeks after e...

Embodiment 2

[0063] Using the optimized vector combination, blood cell reprogramming and high-efficiency AAVS1 locus gene editing can be realized. Targeting the AAVS1 locus, an sgRNA was designed between exon 1 and exon 2 to generate a DSB. Double-cut pDonor includes LHA sequence, an EF1-GFP-Wpre-polyA sequence and RHA sequence ( Figure 4 ). After 2 weeks of electroporation, the reprogramming efficiency can be calculated by counting iPSCs, and the gene editing efficiency can also be obtained by counting the proportion of GFP fluorescence positive cells. We compared 3 vector combinations, including combination 1: OS 2ug+M 1ug+K 1ug+B 0.5ug+Cas9 3ug+sgRNA 1ug+pDonor 2ug; combination 2: OS 2ug+M 1ug+B 0.5ug+Cas9- K 4ug+sgRNA 1ug+pDonor 2ug; combination 3: OS 2ug+M 1ug+B 0.5ug+Cas9-K 4ug+sgRNA 1ug+pDonor 2ug+SV40LT 1ug. After clone counting, we found that combination 1 had the highest reprogramming efficiency, combination 2 was the lowest, and combination 3 was in the middle ( Figure 5 )...

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Abstract

The present invention relates to a method for high-efficiency blood cell reprogramming and simultaneously realizing gene editing, the method comprising: introducing a carrier combination into blood cells by electrotransfection, and reprogramming blood cells into iPSCs; the carrier combination includes pEV-OCT4-E2A-SOX2(OS), pEV-BCL-XL(B), pEV-MYC(M), pEV-Cas9-E2A-KLF4(Cas9-K). According to different applications and expected purpose requirements, the present invention can select an appropriate combination of vectors to realize reprogramming of blood cells into iPSCs at different reprogramming efficiencies and at the same time realize high-efficiency gene editing.

Description

technical field [0001] The invention belongs to the field of cell reprogramming and gene editing, and specifically relates to the technology and application of using Episomal vector to realize high-efficiency blood cell reprogramming and high-efficiency gene editing at the same time. Background technique [0002] Induced pluripotent stem cells (iPSCs) are considered to be a revolutionary cell resource in the fields of stem cell therapy, drug discovery and disease modeling. Blood cells are currently widely used in clinical diagnosis because of their advantages of easy acquisition and low trauma. Previous literature reports expressed Yamanaka factors using retroviral vectors [0003] (OCT4, SOX2, MYC and KLF4) can achieve reprogramming in a variety of cells including blood cells. For cell therapy and related research, non-integrated iPSCs without exogenous genes are more needed. Episomal vectors can be used to reprogram blood cells into non-integrated iPSCs. The most commo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/113
CPCC12N5/0696C12N15/113C12N2310/10C12N2501/603C12N2501/604C12N2501/606C12N2501/602C12N2506/11
Inventor 程涛张孝兵温伟张健萍许静
Owner INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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