Plant large fragment DNA site-directed insertion vector, method and application thereof

By using a site-directed insertion vector containing fusion protein A and donor DNA in plant genome editing, the problem of low insertion efficiency of large exogenous DNA fragments has been solved, enabling precise insertion of kb-level fragments in plants such as tobacco and rice, thus improving insertion efficiency and applicability.

CN122189074APending Publication Date: 2026-06-12SHANXI UNIV
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
SHANXI UNIV
Filing Date
2026-04-29
Publication Date
2026-06-12

AI Technical Summary

⚠Technical Problem

In existing plant genome editing technologies, the precise site-directed insertion efficiency of large exogenous DNA fragments is low, the site dependence is strong, and the local effective concentration of the donor template near the DNA double-strand break site is insufficient, resulting in low insertion efficiency and insufficient reproducibility.

⚗Method used

A plant-derived large-fragment DNA insertion vector is employed, comprising a fusion protein A (composed of a Cas9 protein or a variant thereof linked to an integration-deficient piggyBac variant) and donor DNA. The integration-deficient piggyBac variant retains the ability to recognize terminal repeat sequences and, combined with the repair pathway-biased Cas9 variant vCas9, achieves physical co-localization of donor DNA with target site DNA double-strand breaks, increasing the local effective concentration of the donor DNA near the break site. Furthermore, homologous targeted repair is promoted through transcriptional coupling elements, thereby improving the efficiency of HDR-mediated precise insertion of large-fragment DNA.

🎯Benefits of technology

Precise insertion of kb-level fragments has been achieved in plants such as tobacco and rice, improving cross-site and cross-species applicability, enhancing the accuracy and design flexibility of insertion sites, and improving the site-specific insertion efficiency of large DNA fragments.

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Abstract

This invention belongs to the field of plant genetic engineering and gene editing technology, specifically relating to a plant large-fragment DNA site-directed insertion vector, method, and application. The vector comprises: fusion protein A, which is formed by linking Cas9 protein or its variant with an integration-deficient piggyBac variant; sgRNA; and donor DNA; wherein the integration-deficient piggyBac variant is ipB7. RKD The variant; the donor DNA includes a left homologous arm, a target insertion fragment, and a right homologous arm, and the donor DNA also includes left- and / or right-terminal repeat sequences of piggyBac for recognition by the integration-deficient piggyBac variant. This invention significantly improves the homologous-directed repair insertion efficiency of large DNA fragments in plants by physically anchoring the donor DNA near Cas9-induced DNA double-strand breaks and combining it with a repair pathway-biased Cas9 variant, achieving precise site-specific integration of the target fragment at endogenous sites. This invention is applicable to both dicotyledonous and monocotyledonous plants and can be used for endogenous gene markers, reporter gene introduction, functional module installation, and molecular breeding.
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