Chimeric RNA for precise genomic insertion

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Solution Overview

Problem

Current methods for targeted transgene insertion and allelic replacement in cells face challenges with low frequency of precise insertion and high occurrence of non-homologous end joining, leading to unpredictable and imprecise junction sequences.

Innovation Solution

The use of an engineered chimeric RNA comprising a guide RNA and a bait RNA, which guides a site-directed modifying polypeptide to a specific genomic locus, facilitating targeted integration of a transgene or allelic replacement by enhancing the precision and frequency of homologous recombination.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If site-directed nucleases are used for targeted insertion, then targeted mutagenesis and deletion can be achieved, but the frequency of precise targeted insertion remains low and NHEJ process causes unpredictable junction sequences

Engineering Contradiction:

Improveprecision of targeted insertionVSAvoidfrequency of precise targeted insertion

Core Design Contradiction:

Manufacturing precisionVSProductivity

Solution Approach 1:

The guide RNA is segmented into two separate functional domains: crRNA (for target recognition and binding to Cas9) and bait RNA (for recruiting donor DNA). This segmentation allows independent optimization of each function, improving both precision of targeting and frequency of precise insertion by separating the nuclease guidance function from the donor recruitment function.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The bait RNA acts as an intermediary molecule that bridges the guide RNA-Cas9 complex and the donor DNA. It mediates the recruitment of donor DNA to the targeted genomic locus, facilitating homologous recombination and increasing the frequency of precise targeted insertion while maintaining junction sequence predictability.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Adaptability or versatility

If NHEJ process is used for end repair, then multiple donor DNA fragments can be inserted in multiple orientations, but the junction sequence becomes unpredictable and imprecise

Engineering Contradiction:

Improveflexibility in donor DNA insertionVSAvoidprecision of junction sequence

Core Design Contradiction:

Adaptability or versatilityVSManufacturing precision

Solution Approach 1:

The invention converts the harmful effect of NHEJ (imprecise junction formation) into a beneficial outcome by using the bait RNA to actively recruit donor DNA with specific sequences. The bait RNA's complementarity to the donor DNA ensures precise junction sequences while still allowing flexible donor DNA insertion, effectively converting the randomness of NHEJ into controlled precision.

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

Solution Approach 2:

The invention changes the key parameter of donor DNA recruitment from passive NHEJ-mediated random joining to active bait RNA-mediated specific binding. By altering the recruitment mechanism parameter, the system achieves both adaptability in donor DNA insertion and precision in junction sequence formation through complementary base pairing.

Inventive Principle:

Principle #35Parameter changes

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

This approach significantly improves the targeted integration efficiency of transgenes and allelic replacement, achieving higher precision and frequency compared to methods without the chimeric RNA, thereby overcoming the limitations of existing technologies.

Implementation Method 1

The guide RNA comprises a crRNA segment, which comprising a guide sequence capable of hybridizing to a target sequence

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

The bait RNA segment comprises at least 8 nucleotides, wherein the nucleic acid sequence of the bait segment is complementary to at least 8 nucleic acids of a donor DNA molecule and is capable of hybridizing to the donor DNA molecule

Methodology Applied

Scientific EffectHybridization:

Implementation Method 3

a site-directed modifying polypeptide capable of site-directed cleavage at the target genomic site

Methodology Applied

Scientific EffectNuclease cleavage: Enzyme

Data Source

PatentUS20250129367A1Methods and compositions for targeted genomic insertion

Publication Date: 2025.04.24 SYNGENTA CROP PROTECITON AG

AI summary

The present invention relates to methods and compositions for chimeric RNA comprising a guide RNA and bait RNA for modifying a target site in the genome of a cell. Such modifications include integration of a transgene and allelic mutations and modifications of native genes. Also provided are plants comprising a modified nucleic acid sequence compared to the native gene integrated into a targeted genomic site in the plant genome.