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A composition, method and application for nucleotide sequence modification

A nucleotide sequence and composition technology, which is applied in the field of nucleotide sequence modification composition, can solve problems such as cumbersome methods, inability to target CBE, and limit the application range of single-base gene editing systems

Active Publication Date: 2020-10-30
EAST CHINA NORMAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for bases other than 3-8 within 20bp of the target, it cannot be targeted by spCas9-mediated CBE
The recently reported BE-Plus recruits multiple cytosine deaminases fused with Scfv through 10×GCN4, and can also target cytosine at positions 4-16, and its efficient working window is C at positions 7-13, realizing It realizes the translation of its efficient working window, but its method is relatively cumbersome
This greatly limits the scope of application of the single-base gene editing system

Method used

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  • A composition, method and application for nucleotide sequence modification
  • A composition, method and application for nucleotide sequence modification
  • A composition, method and application for nucleotide sequence modification

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Vector construction

[0046] (1) Construction of the first vector

[0047] The first expression element group on the first vector is such as figure 1 shown ( figure 1 PW-CBE-AID), which includes: cytosine deaminase (AID) expression element, wild-type adenine deaminase (TadA) expression element, mutant adenine deaminase (TadAE59A, which is compared to TadA, whose amino acid residue at position 59 is mutated from E to A) expression element and mutant Cas enzyme (SpCas9n) expression element; each expression element is connected by Linker.

[0048] Wherein, the amino acid sequence of AID is shown in positions 1-182 of SEQ ID NO.6, the amino acid sequence of Linker1 is shown in positions 183-198 of SEQ ID NO.6, and the amino acid sequence of TadA is shown in SEQ ID NO.6 The amino acid sequence of Linker2 is shown in positions 365-396 of SEQ ID NO.6, the amino acid sequence of TadAE59A is shown in positions 397-562 of SEQ ID NO.6, and the amino acid sequence of Linker3 is ...

Embodiment 2

[0056] Validation of the working window for genetic modification of the composition of Example 1

[0057] (1) Download human genes PD-1 and KCNS1 from NCBI, in which PD-1 has designed 4 targets and KCNS1 has designed 1 target (such as Table-1, the underline in the table is PAM), similar to CRISPR / Cas9 target oligo design strategy, the sgRNA uses U6 as the promoter and needs G as the transcription start site, CACC is added to the 5' end of the forward oligo for each target, and the reverse oligo is the complementary strand of the target. AAAC was added at the 5' end (see Table 2).

[0058] Table 1 Target nucleotide sequences on human gene PD-1

[0059]

[0060]

[0061] Table 2 Sequences of forward and reverse oligos for different targets

[0062] target name sequence (5`-3`) PD-1-sg6-up CACCGTCCAGGCATGCAGATCCCCAC PD-1-sg6-dn AAACGTGGGATCTGCATGCCTGGAC PD-1-sg7-up CACCGTGCAGATCCCCACAGGCGCCC PD-1-sg7-dn AAACGGGCGCCTGTGGGATCTGCAC ...

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Abstract

The invention discloses a composition for modifying a nucleotide sequence, a method and application, and relates to the technical field of gene editing. The composition comprises a first vector and asecond vector, wherein the first vector is provided with the following expression elements, such as a cytosine deaminase expression element, an adenine deaminase expression element and a mutant Cas enzyme expression element; the second vector is provided with the following expression elements, such as a gRNA (guide ribonucleic acid) expression element and a uracil glycosidase inhibitor expressionelement. The composition is used for modifying the C bases at the 1st to 16th sites at the upstream of PAM (protospacer adjacent motif) of the target nucleotide sequence, so that the transformation ofC / G to T / A is realized. Compared with the existing gene editing technique, the composition and the method have the advantages that the working window is broader; the DSB (double-strand break) and indels (insertion / deletion mutations) are not introduced, the off-target effect is very low, the safety is higher, and the like.

Description

technical field [0001] The present invention relates to the technical field of gene editing, in particular, to a composition, method and application for nucleotide sequence modification. Background technique [0002] Since 2013, the new generation of gene editing technology represented by CRISPR / Cas9 has entered various experiments in the field of biology, which is changing the traditional means of gene manipulation. [0003] In April 2016, the laboratory of David R Liu first reported the single-base gene editing technology (cytosine base editor, CBE) based on rat cytosine deaminase (Apobec1) CRISPR / Cas9 fusion (including BE1, BE2, BE3) Genome-directed editing of single-base C / G to T / A transitions on the genome. Among them, BE3 is widely used in gene mutation or repair of genome due to its high efficiency, making disease animal models, gene therapy, gene function screening, etc. The classic CBE system (referring to BE3) is based on spCas9 transformation. In addition to rec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/10
CPCC12N15/102C12N15/85
Inventor 李大力张晓辉陈亮刘明耀
Owner EAST CHINA NORMAL UNIV
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