Preparation and application of induced mutant protein based on activated induced cytidine deaminase

A technology of cytidine deaminase and mutant protein, which is applied in the direction of immunoglobulin, DNA preparation, botanical equipment and methods, etc. It can solve the problems of large molecular weight, high off-target, difficult to transport targeted DNA fragments, etc., and achieve small molecular weight , the effect of high mutation efficiency

Active Publication Date: 2020-08-11
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The mutation efficiency of the existing AID is not high enough; the single base editing system based on the CRISPR / Cas9 system and AID has a large molecular weight, is not easy to transport to the targeted DNA fragment, and limits the transgenic application of some species; the CRISPR / Cas9 system Has a high off-target (Off Target) effect

Method used

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  • Preparation and application of induced mutant protein based on activated induced cytidine deaminase
  • Preparation and application of induced mutant protein based on activated induced cytidine deaminase
  • Preparation and application of induced mutant protein based on activated induced cytidine deaminase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Design and optimization of activation-inducible cytidine deaminase (AID)

[0042] 1. Screening for AIDs with high mutation rate

[0043] Obtain the sequence of the deaminase gene family by sequence alignment, and divide it into several categories according to its structure on the evolutionary tree ( figure 1 ). And select representative deaminases on each major branch for downstream screening experiments.

[0044]The deaminase gene was integrated into an inducible expression vector (pGA) after codon optimization, and the mutation efficiency was determined in the yeast platform. Results from figure 2 It can be seen that GIL104 yeasts expressing different deaminases all had resistant clones on the SC-Arg- / CAN+ plate, indicating that the deaminase genes were successfully expressed. In the experiment, each sample has 36 independent plate repetitions, and the number of yeast cells in each plate repetition is 8×10 6 , In addition, due to the large number of ...

Embodiment 2D

[0051] Example 2 Screening and optimization of DNA-specific binding proteins

[0052] The reason why AID protein can directional induce hypermutation of specific locus in vivo is because there are a large number of cofactors and complex spatiotemporal regulation mechanism. If there is only a single overexpression of AID protein, on the one hand, the mutation rate cannot reach a high level, and on the other hand, random mutations will occur at all positions in the genome, causing mutation burden (mutation burden). Therefore, the work of the "targeted recording" system also requires the assistance of DNA-specific binding proteins. "Mutator" (mutator) acts on a specific region of the genome through the traction of DNA-specific binding proteins. In the description below, "DNA-specific binding protein" is simply referred to as "targeter".

[0053] Currently widely used DNA-specific binding proteins can be divided into three categories: zinc finger proteins, TALENs proteins, and C...

Embodiment 3

[0055] Example 3 Preparation of single-base site-directed editing protein (HBE) (yeast system)

[0056] Through the screening and optimization of the "mutator" and "targeter", the two core parts of the "recording protein" in the targeted recording system were preliminarily obtained. The prototype of the recording protein can be obtained by linking the two parts with a flexible peptide chain. Then, through the overall optimization of the fusion protein and the addition of other enhancement elements, a "high-efficiency single-base fixed-point editing protein" (High-efficiency BaseEditor, HBE for short) can be obtained.

[0057] Further optimization and screening of the AID protein: using the AID5 protein in Example 1 as a template, a mutant library of the AID protein was obtained by error-prone PCR. In the final library, each molecule contained ~4 base substitution mutations. After gel recovery, the AID library was constructed into the pGA inducible vector with a library size o...

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Abstract

The invention provides a mutant protein based on activated induced cytidine deaminase. The mutant protein is obtained by mutating hsAID as follows: T82I, K10E, K34E, E156G, 181 *, S38, H130, V152, R174 and T100. The invention also provides a single base site-specific editing protein. The protein comprises the mutant protein based on activated induced cytidine deaminase and a DNA specific binding protein, wherein the mutant protein based on activated induced cytidine deaminase and the DNA specific binding protein are sequentially connected through a connecting sequence. The invention also provides a single base site-specific editing system. The system comprises the single base site-specific editing protein and a targeted high mutation sequence. Compared with the existing single base editingsystem based on activated induced cytidine deaminase (AID), the induced mutant protein system provided by the invention has the advantages of smaller molecular weight and higher mutation efficiency.

Description

technical field [0001] The invention belongs to the technical field of gene editing, and in particular relates to the preparation and use of an inducible mutant protein based on activation-inducible cytidine deaminase. Background technique [0002] Inducible cytidine deaminase (Activationinduced cytosine deaminase, AICDA, or AID) is a class of DNA editing enzymes, in somatic hypermutation (SHM), gene conversion (gene conversion) and class switch recombination in B lymphocytes ( Class Switch Recombination (CSR) plays an important role in deamination of Ig variable region (V) and Ig switch region (S) DNA, greatly increasing the diversity of the immune repertoire. [0003] It works roughly as follows: first, it replaces cytosine (C) with uracil (U), and then in the next round of DNA replication, this uracil (U) is converted to thymine (T). If the intracellular repair mechanism detects the presence of uracil (U) in DNA, there is also a certain probability that it will trigger t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/78C12N15/55C12N15/62C07K19/00C12N15/10
CPCC12N9/78C12N15/102C12Y305/04005C07K2319/80C07K2319/09C12N2310/20C07K16/00
Inventor 贺雄雷刘黎叶畅刘科辉邓善俊
Owner SUN YAT SEN UNIV
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