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Fusion protein for enhancing gene editing, and application of fusion protein
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A technology of fusion protein and gene editing, applied in the field of fusion protein for enhanced gene editing, which can solve the problems of low editing efficiency, off-target, limited target selection, etc.
Active Publication Date: 2020-09-29
EAST CHINA NORMAL UNIV +1
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[0003] Although CRISPR / Cas9 technology has extremely powerful functions, it also has its disadvantages, such as: 1. Off-target problems; 2. The limitation of PAM leads to limited target selection; 3. The editing efficiency of some new tools is generally low ( XCas9 and SpCas0-NG), and the current optimization and transformation of gene editing tools is mainly based on the improvement of the accuracy of the tool and the enhancement of the targeting range, and there is no extensive improvement of various gene editing tools for the gene editing tools themselves. Methods
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Embodiment 1
[0196] Example 1 Screening Enhanced Gene Editing Tool
[0197] Synthesize different types of double-stranded DNA binding domains (HMG-D and Sac7d), respectively, by designing 5 different length linkers (L1, L2, L3, L4 and L5) (Table 1), and fusion at the N-terminal of Cas9 and C-terminal, compared on two endogenous targets (VEGF and HBG1 / 2), according to the statistics of the results, the enhanced gene editing tools obtained by this series of optimizations are scored (in the cases where the effects are all very good Based on the relative score) (bad: A; not good: AA; better: AAA; good: AAAA; very good: AAAAA), the results are as follows: (Note: C means Cas9; L1-L5 means different linkers ; H indicates HMG-D domain; S indicates sac7d.)
[0205] Example 2 Improves the gene editing efficiency of SpCas9
[0206] The obtained enhanced gene editing tool (that is, the fusion protein of the present invention, such as HMG-D-L4-SpCas9) is further compared in terms of effects on more endogenous targets, and 293T cells are transfected by an equimolar ratio , it was found that compared with SpCas9 in the compared targets, the editing efficiency of the fusion protein of the present invention was increased by more than 20% (or 60%, or 80%), up to 2 times ( figure 2 ).
Embodiment 3
[0207] Example 3 Improves the gene editing efficiency of Cas9 (such as: SaCas9) derived from other species
[0208] By constructing the HMG-D-L4-SaCas9 expression vector and transfecting 293T cells at an equimolar ratio for the endogenous target, it was found that the editing efficiency of SaCas9 after the fusion of HMG-D also had a similar effect. ( image 3 )
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Abstract
The invention relates to a fusion protein for enhancing gene editing, and an application of the fusion protein. Specifically, the invention provides an enhanced fusion protein. Compared with wild-typegene editing proteins, the enhanced fusion protein of the invention can significantly improve the gene editing efficiency in vivo or in vitro.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a fusion protein that enhances gene editing and its application. Background technique [0002] Gene editing technology is a technology that artificially achieves double-strand DNA breaks and uses the repair mechanism of double-strand DNA breaks to achieve genetic manipulation. The existing gene editing technologies include ZFN, TALEN and CRISPR / Cas9 technology, among which CRISPR / Cas9 technology is the most widely used. CRISPR / Csa9 technology is an acquired immune mechanism derived from bacteria or archaea. It uses a single-stranded guide RNA (sgRNA) and Csa9 protein to generate DNA double-strand breaks at specific positions in the genome, and then through endogenous non-identical Original end joining (NHEJ) or homologous recombination (HDR) repair mechanism to achieve the knockout of the target gene or the insertion of a specific gene or fragment. [0003] Although CRISPR / Cas9 tec...
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