System and Method of Induced Mutant Protein Based on Activation-induced Cytidine Deaminase
a technology of activation-induced cytidine deaminase and system, which is applied in the field of gene editing, can solve the problems of limited application of homology-directed repair technology, limited efficiency of precise gene editing mediated by homology-directed repair, and limited wide application of this technology, so as to achieve small molecular weight and higher mutation efficiency
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example 1
Design and Optimization of Activation-Induced Cytidine Deaminase (AID)
[0043]1. Screen AID with High Mutation Rate
[0044]The sequence of the deaminase gene family is obtained through sequence alignment, and classified into several major categories according to its structure on the evolutionary tree (FIG. 1). And select representative deaminase from each main branch for downstream screening experiments.
[0045]The deaminase gene is integrated into the induction expression vector (pGA) after codon optimization, and the mutation efficiency is determined in the yeast platform. The results can be seen in FIG. 2 that GIL104 yeast expressing different deaminases all appeared resistant clones on the SC-Arg− / CAN+ plate, indicating the successful expression of the deaminase gene. In the experiment, each sample has 36 independent replicates. The number of yeast cells in each TLC plate replicate is 8×106. In addition, due to the large number of clones, the hsAID sample was diluted 10 times before s...
example 2
Screening and Optimization of DNA-Specific Binding Proteins
[0052]The reason why AID protein can induce high mutations at specific locus in vivo is due to a large number of cofactors and complex time-conditioning control mechanisms. If it is only a simple overexpression of AID protein, the mutation rate cannot reach a high level on the one hand, on the other hand, mutations will be randomly generated in all positions of the genome, thus triggering mutation burden. Therefore, the job of the “targeted recording” system requires the assistance of DNA-specific binding proteins. The “mutator” is pulled by a DNA-specific binding protein to target specific regions in the genome. In the following description, “DNA specific binding protein” is referred to as “targeter” for abbreviation.
[0053]Currently widely used DNA-specific binding proteins can be classified into three categories: zinc finger proteins, TALENs proteins, and CRISPR / Cas proteins. Wherein, zinc finger proteins have short recogn...
example 3 preparation
of High-Efficiency Base Editor (HBE) (Yeast System)
[0055]Through the screening and optimization of “mutator” and “targeter”, the two core parts of the “recording protein” in the target recording system were initially obtained. Use a flexible peptide chain to connect the two parts to obtain the prototype of the recorded protein. Then, by overall optimizing the fusion protein, and adding other enhancing elements, there obtained the “High-efficiency Base Editor (HBE)”.
[0056]Further optimizing and screening the AID protein: Using the AID5 protein in Example 1 as a template, a mutation library of the AID protein was obtained by error-prone PCR. In the final library, each molecule contained about 4 base substitution mutations. After gel recovery, the AID library was constructed into the pGA induction vector with a library size of about 105. The plasmid library was transformed into GIL104 yeast strain by lithium acetate transformation method, and positive clones were screened on SC-Leu− / GL...
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