Single-base editing tool TaC9-ABE and application thereof

Abstract

The invention provides a single-base editing tool TaC9-ABE and application thereof. The single-base editing tool TaC9-ABE comprises a first vector for expressing a TALE recognition protein, wild adenosine deaminase and mutant adenosine deaminase; and a second vector for expressing SgRNA and nSpCas9 proteins. The single-base editing tool TaC9-ABE is a double-component gene editor and has the characteristics of high efficiency and safety. Compared with a traditional ABE7.10 system, the editing efficiency of the double-component editor TaC9-ABE is similar to or even higher than that of the traditional ABE7.10 system. A single component in the double-component editor completely has no editing activity at a target site, and an editing phenomenon is not found in a predicted off-target area during simultaneous transfection of the two components, so that by the double-component editor, the recognition specificity of the target site is improved, and the DNA off-target problem caused by Cas9 non-specific binding is reduced.

Description

technical field [0001] The invention belongs to the technical field of gene editing, and in particular relates to a single base editing tool TaC9-ABE and its application. Background technique [0002] Nucleotide editing targeting the human genome has high application value in scientific research and clinical treatment. At present, the most widely used gene editing method is CRISPR / Cas9 technology, which can edit genomes in various biological cells including mammals, but due to the generation of double-strand breaks (DSBs), it will pass error-prone non-identical Source-end joining (NHEJ) causes random insertions and deletions, while homology-directed repair (HDR) of the target is less efficient at the target site, so CRISPR / Cas9 technology has certain problems in terms of clinical application. [0003] At the same time, with the development of the Human Genome Project, it has been confirmed through big data analysis that about 80% of disease-related gene variations are point...

AI Technical Summary

Problems solved by technology

[0006] However, as the research progressed, the researchers found that the off-target effects of single base editing tools were obvious

In March 2019, through whole-genome sequencing analysis, researchers confirmed in mice and rice that, in addition to the traditional Cas9 non-specific binding DNA off-target risk, the CBE system also has a Cas9-independent DNA off-target risk, while Cas9-independent DNA off-target effects of the ABE system are relatively low

[0007] In April 2019, a research paper by J. Keith Joung's team showed that CBE and ABE single-base editing can not only cause DNA mutations, but also lead to a large number of RNA editing

[0011] Although the current research has basically solved the problem of Cas9-independent DNA and RNA off-target in single base editing technology from different directions, there is still no good solution to the problem of DNA off-target based on sgRNA sequence mismatch binding (mismatch tolerance) At present, relatively specific sequences can only be selected through website screening, but due to the limitation of the PAM region and single base editing range, the selection space for the target site is still very limited

At the same time, there are certain differences in the editing efficiency of different sequences. The previous mutation screening based on deaminase has the problems of reduced efficiency and narrowed targeting range. It is still difficult to achieve the goal of safety and efficiency at the same time.

[0012] In summary, although the Cas9-independent DNA off-target and RNA off-target problems have been basically solved by optimizing the deaminase, due to the complexity of the genome, the possibility of highly homologous or even identical sequences to the targeted sgRNA is very high , the resulting DNA off-target problem is likely to cause cytotoxicity, hindering the clinical application of single base editing tools

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