阅读说明：本技术 基于激活诱导性胞苷脱氨酶的诱导突变蛋白的制备和用途 (Preparation and application of induced mutant protein based on activation-induced cytidine deaminase ) 是由 贺雄雷 刘黎 叶畅 刘科辉 邓善俊 于 2020-04-13 设计创作，主要内容包括：本发明提供了一种激活诱导性胞苷脱氨酶的突变蛋白,其是对hsAID进行了如下突变：T82I,K10E,K34E,E156G,181*,S38,H130,V152,R174,T100。本发明还提供了一种单碱基定点编辑蛋白,所述蛋白包括本发明的激活诱导性胞苷脱氨酶的突变蛋白和DNA特异结合蛋白,所述激活诱导性胞苷脱氨酶的突变蛋白和DNA特异结合蛋白经连接序列依次相连。本发明还提供了一种单碱基定点编辑系统,所述系统包括所述的单碱基定点编辑蛋白和靶向高突变序列。本发明所述的诱导突变蛋白系统相比现有的基于激活诱导性胞苷脱氨酶(AID)的单碱基编辑系统,分子量更小,突变效率更高。(The invention provides a mutant protein for activating induced cytidine deaminase, which carries out the following mutations on hsAID: T82I, K10E, K34E, E156G, 181, S38, H130, V152, R174, T100. The invention also provides a single-base fixed-point editing protein, which comprises the mutant protein of the activation-induced cytidine deaminase and the DNA specific binding protein, wherein the mutant protein of the activation-induced cytidine deaminase and the DNA specific binding protein are sequentially connected through a connecting sequence. The invention also provides a single-base fixed-point editing system, which comprises the single-base fixed-point editing protein and a targeted high-mutation sequence. Compared with the existing single base editing system based on activation induced cytidine deaminase (AID), the induced mutein system provided by the invention has the advantages of smaller molecular weight and higher mutation efficiency.)

1. A mutein that activates induced cytidine deaminase wherein hsAID is mutated as follows: T82I, K10E, K34E, E156G, 181, S38, H130, V152, R174, T100; the nucleotide sequence of the mutant protein is shown as SEQ ID NO. 1.

2. A single-base site-directed editing protein comprising the induced cytidine deaminase-activating mutein of claim 1 or 2, a DNA-specific binding protein, and a nuclear localization signal, wherein the induced cytidine deaminase-activating mutein and the DNA-specific binding protein are linked in sequence by a linker sequence, and the nuclear localization signal is located C-terminal to the single-base site-directed editing protein.

3. The single base site-directed editing protein of claim 2, wherein the DNA-specific binding protein is a homing endonuclease; the nucleotide sequence of the DNA specific binding protein is shown as SEQ ID NO. 2.

4. The single base site-directed editing protein of claim 2 or 3 further comprising a UGI protein domain, wherein the UGI protein domain is located after the DNA specific binding protein and before the nuclear localization signal.

5. A single-base fixed-point editing protein suitable for a yeast system is characterized in that the nucleotide sequence is shown as SEQ ID NO. 3.

6. A single-base fixed-point editing protein suitable for a drosophila system is characterized in that the nucleotide sequence is shown as SEQ ID NO. 4.

7. A single-base fixed-point editing protein suitable for zebra fish and mouse systems is characterized in that the nucleotide sequence is shown as SEQ ID NO. 5.

8. A single base site-directed editing system, which comprises the single base site-directed editing protein according to any one of claims 3 to 8 and a targeted high mutation sequence.

9. The single base site-directed editing system of claim 8, wherein the targeted hypermutation sequence comprises the nucleotide sequences shown as SEQ ID NOs 6 and 7.

10. Use of the single base site-directed editing system of claim 8 or 9 for gene editing.

Technical Field

The invention belongs to the technical field of gene editing, and particularly relates to preparation and application of induced mutant protein based on activation induced cytidine deaminase.

Background

Induced cytidine deaminase (AICDA, or AID) is a DNA editing enzyme that plays an important role in Somatic Hypermutation (SHM), gene conversion (gene conversion) and Class switching recombination (csr) in B lymphocytes, and by deaminating the DNA of Ig variable region (V) and Ig Switch region (S), the diversity of immune repertoires is greatly increased.

The working principle of the device is as follows: first, it replaces cytosine (C) with uracil (U), which is then converted to thymine (T) in the next round of DNA replication. If the intracellular repair machinery detects the presence of uracil (U) in DNA, there is also a probability that base removal will be triggered, resulting in a mutation of C → G or C → A. DNA deaminase effects the conversion of cytosine to thymine by deamination, thereby initiating a mutation in DNA (C → T).

Efficient gene editing has become possible since the advent of the CRISPR/Cas9 system. Cas9 is targeted to a specific DNA region under the direction of a short RNA molecule (guide RNA, gRNA), at which targeting site the Cas9 endonuclease induces a double strand break, which is then primarily repaired in the form of insertions and deletions (indels) by the homologous recombination repair mechanism (HDR). However, the efficiency of precise gene editing mediated by homologous recombination repair is limited, which limits the wide application of the technology, so that precise gene editing, such as single base change, remains a great challenge to the CRISPR technology.

Later, researchers found that a single-base editing system developed by integrating base deaminases (such as cytidine deaminase APOBEC1 and adenosine deaminase TadA variants) and a CRISPR/Cas system can accurately introduce C/G-T/A and A/T-G/C point mutations without cutting double strands of DNA, thereby realizing efficient and accurate gene editing. This gene editing system is still RNA guided, but it does not cause double strand breaks at the target site. In contrast, cytidine deaminase converts cytosine bases to uridine, which is then repaired by an error-prone mechanism, resulting in various point mutations. Furthermore, the system can also achieve more specific and desirable point mutations, such as C-T or G-A transitions, when the uracil-DNA glycosylase pathway is inhibited. This progress in gene editing is very important because two thirds of human genetic diseases are caused by single base changes. Theoretically, the single-base editing system can be used for treating hundreds of genetic diseases and has great clinical application potential.

The single base editing system CRISPR-Cas9-AID based on B cell specific activation induced cytidine deaminase (AID) is a more important class of single base editing technology.

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 large molecular weight, is not easy to transport to a target DNA fragment, and has limitation on the transgenic application of partial species; the CRISPR/Cas9 system has a higher Off-Target (Off Target) effect.

Disclosure of Invention

The invention mainly solves the technical problem of providing an induced mutant protein system based on activation induced cytidine deaminase (AID) and is named as 'high-efficiency single-base fixed-point editing protein' (HBE). The system can specifically target DNA and induce DNA mutation with high efficiency. Meanwhile, the invention also provides preparation and application of the system in mice.

In order to achieve the purpose, the invention adopts the technical scheme that:

in a first aspect the invention provides a mutein that activates induced cytidine deaminase, wherein the mutein is obtained by mutating hsAID as follows: T82I, K10E, K34E, E156G, 181, S38, H130, V152, R174, T100.

Further, the nucleotide sequence of the mutant protein is shown as SEQ ID NO. 1.

In a second aspect, the present invention provides a single-base site-directed editing protein (HBE), which comprises the above-mentioned mutein activating inducible cytidine deaminase, a DNA-specific binding protein, and a nuclear localization signal, wherein the mutein activating inducible cytidine deaminase and the DNA-specific binding protein are sequentially linked via a linker sequence, and the nuclear localization signal is located at the C-terminus of the single-base site-directed editing protein.

Further, the DNA specific binding protein is a homing endonuclease.

Further, the homing endonucleases include iSCell, iTevl, iSmaMl, piScel, iPoll, piPful, iHmul, iCrel, iCeul, iAnil.

Furthermore, the nucleotide sequence of the DNA specific binding protein is shown as SEQ ID NO. 2.

Further, a UGI protein domain is included, the UGI protein domain being located after the DNA specific binding protein and before the nuclear localization signal.

The key elements AID10, dISceI, UGI and SV40 in the single-base site-directed editing protein (HBE) are connected in series through a connecting sequence to form a protein with a single-base site-directed editing function, and the connecting sequence can be adjusted according to the requirements of an expression system or a host cell. In the embodiment, the invention provides HBE proteins suitable for a yeast system, drosophila, zebrafish and mice, key elements AID10, dISceI, UGI and SV40 in the HBE proteins are consistent, except for the connection sequences, and the sequence for connecting AID10 and dISceI in the yeast system is 6 x (GGGGS); the linker sequence was XTEN, 6 × (GGGGS) in drosophila; the ligation sequences in zebrafish and mouse systems were 6 × (GGGGS), GS-rich-linker and HA. Thus, it was demonstrated that the linker sequence can be regulated according to the expression system. Therefore, in the embodiment, the HBE protein suitable for a yeast system, drosophila melanogaster, zebra fish and mice is obtained, and the nucleotide sequences of the HBE protein are respectively shown in SEQ ID NO. 3-5.

In a third aspect the present invention provides a single base site directed editing system comprising a single base site directed editing protein as described above and a targeted highly mutated sequence.

Furthermore, the nucleotide sequence of the targeted high mutation sequence (HMF) is shown as SEQ ID NO:6 and SEQ ID NO: 7.

In a fourth aspect, the invention provides the use of a single base site-directed editing system in gene editing, particularly the use of single base site-directed mutagenesis.

The invention has the beneficial effects that: compared with the existing single base editing system based on activation induced cytidine deaminase (AID), the induced mutein system provided by the invention has the advantages of smaller molecular weight and higher mutation efficiency.

Drawings

FIG. 1 is a schematic representation of a evolutionary tree of the deaminase gene family.

FIG. 2 is a graph showing the results of screening different deaminases by yeast spotting.

FIG. 3 is a graph showing the results of comparison of the mutagenesis efficiencies of different deaminases (the ordinate indicates different deaminases and the abscissa indicates the mutation rate).

FIG. 4 is a graphical representation of the results of screening for variants of hsAID by yeast dot-plate assay.

Fig. 5 is a schematic diagram of the protein mutation site and 3D structure of hsAID mutant AID 5.

Fig. 6 is a graph comparing the mutational efficiencies of AID5 and AID10 by yeast dot-plate experiments.

Fig. 7 is a schematic diagram of the protein mutation site and 3D structure of hsAID mutant AID 10.

FIG. 8 is a graph showing the results of screening for DNA binding proteins (different DNA binding proteins are shown on the ordinate and mutation rates are shown on the abscissa).

FIG. 9 is a diagram showing a single base site-directed editing protein (HBE) sequence map and a protein 3D structure in yeast.

FIG. 10 is a sequence map of HBE protein in Drosophila.

FIG. 11 is a sequence map of HBE protein in zebrafish and mice.

Fig. 12 is a flow chart of the design and optimization of targeted Highly Mutated Sequences (HMFs).

FIG. 13 is a map of the tet-SLOTH system in the mouse H11 transgene locus.

FIG. 14 is a schematic representation of the strain construction of tet-SLOTH mice.

FIG. 15 is a map of the lox-SLOTH system in mouse Rosa26 site.

FIG. 16 is a schematic diagram of strain construction of lox-SLOTH mice.

FIG. 17 is a diagram showing the expression of HBE gene in tet-SLOTH system under Dox-induced conditions.

FIG. 18 is a statistical plot of the number of mutation events on the HMF3k fragment in the mouse system.

Detailed Description

In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments and accompanying drawings.