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Adenine single-base editing product without PAM limitation, method and application
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A single-base, gene-editing technology, applied in the fields of genetic engineering and protein modification, can solve problems such as editing efficiency limitations and achieve good application prospects
Pending Publication Date: 2022-05-31
EAST CHINA NORMAL UNIV +1
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Although the corresponding mutation on the complementary strand of the mutation site is theoretically repairable by ABE (G>A), since the region on the complementary strand including the mutation site (G>A) is not suitable for ABE7. PAM, coupled with the limitation of ABE7.10 editing efficiency in hematopoietic stem / progenitor cells, has been unable to achieve effective repair of this pathogenic mutation by single base editing
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Embodiment 1
[0072] Example 1. Guided by NGG PAM sgRNA and non-NGG PAM sgRNA respectively in healthy human hematopoietic stem / progenitor cells to achieve efficient introduction of A>G substitution at the target site (taking the electroporation delivery method as an example)
[0073] The healthy human hematopoietic stem / progenitor cells used in this example come from the peripheral blood of healthy people.
[0074] 1. Design of sgRNA
[0075] Design a non-NGG PAM sgRNA at the NACT-2 site in the intergenic region of the normal site, numbered sgNACT-2 (PAM is GGCT), and its targeting sequence is:
[0076] sgNACT-2: (SEQ ID No. 5);
[0077] Design an NGG PAM sgRNA in the normal site BCL11A gene enhancer region, numbered sg1620 (PAM is GGG), and its targeting sequence is:
[0078] sg1620: (SEQ ID No. 6);
[0079] Among them, the above two target sequences are both 20bp in length, and A in the ABE-SPRY editing window is located at the bold horizontal line mark in the above sequence.
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Embodiment 2
[0109] Example 2. Repair of disease-causing sites in β-thalassemia IVS2-654 C>T-deficient hematopoietic stem / progenitor cells (taking electroporation as an example)
[0110] The β-thalassemia IVS2-654 C>T-deficient hematopoietic stem / progenitor cells used in this example came from patients heterozygous for β-thalassemia IVS2-654.
[0111] 1. Design of sgRNA
[0112] Design non-NGG PAM sgRNAs within 20 bp including the abnormal splicing mutation IVS2-654 C>T site, numbered IVS2-654-sg1 to IVS2-654-sg5, and the target sequences are:
[0113] IVS2-654-sg1: (SEQ ID No.7, PAM is TCA);
[0114] IVS2-654-sg2: (SEQ ID No.8, PAM is ATC);
[0115] IVS2-654-sg3: (SEQ ID No.9, PAM is TAT);
[0116] IVS2-654-sg4: (SEQ ID No.10, PAM is TTA);
[0117] IVS2-654-sg5: (SEQ ID No.11, PAM is ATT);
[0118] Among them, the above five targeting sequences are all 20bp in length, and the IVS2-654 C>T mutation site (the complementary strand is A, located at the bold horizontal line mark...
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Abstract
The invention discloses a PAM limitation-free adenine single base editing product, a PAM limitation-free adenine single base editing method and an application of the PAM limitation-free adenine single base editing product. The product comprises a fusion protein, the fusion protein comprises the following two parts: adenine nucleoside deaminase and endonuclease, the amino acid sequence of the adenine nucleoside deaminase comprises a sequence as shown in SEQ ID No.1, and the amino acid sequence of the endonuclease comprises a sequence as shown in SEQ ID No.2. The product can realize the introduction of Agt without PAM limitation into the genes of cells, especially hematopoietic stem / progenitor cells; g, replacement editing is carried out, the edited autologous hematopoietic stem cells of the patient are transfused back, and the purpose of thoroughly curing diseases in a long-acting mode can be achieved. The product is applied to repair of beta-thalassemia (thalassemia) related mutation IVS2-654 Cgt; the beta-globingene editing efficiency is high, the expression level of the beta-globingene can be remarkably and effectively improved, and the beta-globingene editing method has the application potential in clinical treatment of beta-anemia.
Description
technical field [0001] The present invention relates to the technical field of genetic engineering and protein transformation, in particular to a product, method and application of adenine single base editing without PAM restriction. Background technique [0002] The CRISPR / Cas9 gene editing system can cut at a specific part of DNA to generate a DNA double-strand break (DSB), and there are two repair mechanisms in the cell, non-homologous end joining and homologous recombination repair, so as to realize genome editing. But the original CRISPR technology involves DNA double-strand breaks, which pose potential risks. The development of a single-base gene editing system can achieve precise modification of specific genetic loci without causing DNA double-strand breaks. The base editor in the single-base gene editing system is mainly composed of two parts: Cas protein and DNA modificationenzyme. So far, two types of base editors have been developed: cytosine base editor (CBE),...
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