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Inductive base editing system and application thereof

A base editing and inducible technology, applied in the field of genetic engineering, can solve problems such as amplification of off-target consequences, and achieve the effect of shortening editing time and reducing off-target editing

Active Publication Date: 2021-06-01
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Considering that AAV is currently one of the most effective in vivo delivery vectors, AAV injection will cause long-term expression of transgenes in vivo, and the use of this vector to deliver base editors will undoubtedly amplify off-target consequences

Method used

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  • Inductive base editing system and application thereof
  • Inductive base editing system and application thereof
  • Inductive base editing system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 constructs split-A3A plasmid

[0033] The split-A3A plasmid was constructed on the basis of A3A-BE3 to form a series of N-terminal split-A3A and its corresponding C-terminal split-BE3-A3As ( figure 1 b). Such as figure 1 As shown in c, four groups of split-A3A-BE3 (sA3A-BE3) were constructed and named after their amino acid splitting sites, namely sA3A-BE3-44, sA3A-BE3-85, sA3A-BE3-118 and sA3A- BE3 is -147 respectively.

[0034] figure 1 It is the design of inducible split-A3A-BE3; among them, panel a shows the schematic diagram of the reassembly of inducible split-A3A-BE3, and rapamycin induces the dimerization of FRB and FKBP fused to the N-terminus and C-terminus of A3A, respectively To form a heterodimer, thereby completing the assembly of a functional A3A-BE3 base editor. Figure b is a schematic diagram of the structure of A3A (PDB: 5keg). The four unstructured loops opposite the ssDNA-A3A binding interface are candidate cleavage sites, represen...

Embodiment 2

[0035] Example 2 split-A3A base editing

[0036] 1. Detection of inducible base editing activity of sA3A-BE3

[0037] Each pair of split-A3A-BE3 editors was separately co-transfected with sgRNAs for each of the 3 endogenous targets into HEK293T cells. Such as figure 2 In a, all sA3A-BE3s exhibit inducible editing activity, albeit with different potentials. Among these sA3A-BE3, sA3A-BE3-44 edited most efficiently at all 3 target sites when induced with 200 nM rapamycin, a concentration frequently used in the split system of FRB / FRBP. However, its activity remained essentially unchanged under uninduced conditions. On average, uninduced sA3A-BE3-44 exhibited comparable activity to full-length A3A-BE3 ( figure 2 b). Transfection of the C-terminal portion of sA3A-BE3-44 did not produce any detectable editing, suggesting that the background editing activity of sA3A-BE3-44 was not due to the residual deamination activity of C-terminal A3A, but probably due to the autologous d...

Embodiment 3

[0042] Further characterization of embodiment 3 split-A3A

[0043] To examine whether the cleavage of A3A at amino acid 85 changes the base editing mode of A3A-BE3, including editing window, sequence preference and editing purity of C→T.

[0044] To determine the editing window, 3 targets with multiple cytosines within the editing window were selected ( Figure 4 a). Such as Figure 4 a, compared with full-length A3A-BE3, sA3A-BE3-85 does not significantly shift the position of the editing window or affect the width of the editing window. We then tested whether sA3A-BE3-85 has a different sequence preference than full-length A3A-BE3, as previous studies have shown that the unstructured loop between deaminase helices plays an important role in substrate recognition (Salter et al. 2016). The nine targets were divided into four groups according to the NC motif (GC, CC, AC and TC), and the sequence preference of sA3A-BE3-85 and full-length A3A-BE3 was compared ( Figure 4 b)....

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Abstract

The invention discloses an inducible base editing system and an application thereof. The base editing system is composed of inducible human source cytosine deaminase (A3A) and SpCas9 with single nick activity; the deaminase can be split on the basis of potential amino acid sites; rapamycin interacts with the N end and the C end of the amino acid resolution site through FRB / FKBP to recombine split deaminase, and basic group editing is completed through induction. According to the method, the base editing activity is controlled by constructing the inducible base editing system, so that off-target editing is reduced; meanwhile, the splitting design in the application does not reduce target editing, so that the splitting design can be used as a compensation strategy of protein mutation, and can be used in combination with the mutated deaminase to further reduce off-target editing.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an inducible base editing system and its application. Background technique [0002] Base editing is a genome editing strategy that directly converts specific individual nucleotides in genomic DNA (Rees and Liu 2018). Currently, there are two types of base editors, cytosine base editors (CBE) and adenine base editors (ABE), both of which consist of a deaminase enzyme with a catalytically defective Cas9 protein that catalyzes Deamination of cytosine or adenine. Under the guidance of sgRNA, the base editor can convert C-G base pairs to T-A base pairs, or convert A-T base pairs to G-C base pairs within a few base pairs of a specific window within the target site. Because it is efficient and precise, and does not produce by-products such as insertions or deletions introduced by DNA double-strand breaks (DSBs), this technique was soon widely used in various mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/78C12N9/22C12N15/113
CPCC12N9/78C12N9/22C12N15/113C12Y305/04001C12N2310/20
Inventor 姚少华龙洁
Owner SICHUAN UNIV
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