HIV Reverse Transcriptase Prime Editing for Long Insertions

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Solution Overview

Problem

Conventional prime editing methods using Moloney murine leukemia virus (MMLV) reverse transcriptase struggle with inserting long genetic information due to its monomeric nature, limiting the flexibility and precision of genome editing.

Innovation Solution

Incorporating HIV reverse transcriptase instead of MMLV reverse transcriptase in the prime editor protein complex, which includes a target-specific nuclease like nickase Cas9, enhances the efficiency of prime editing by enabling the insertion of longer genetic information.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Length of moving object

If MMLV reverse transcriptase is used in the prime editor protein complex, then the basic prime editing function is achieved, but the insertion of long genetic information is difficult

Engineering Contradiction:

Improvelength of genetic informationVSAvoidediting efficiency

Core Design Contradiction:

Length of moving objectVSReliability

Solution Approach 1:

The patent changes the parameter of reverse transcriptase from MMLV to HIV, which fundamentally alters the enzyme's properties. HIV reverse transcriptase has different structural and functional characteristics that enable it to process longer genetic information more efficiently, directly resolving the contradiction between inserting long genetic information and maintaining editing efficiency

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent uses HIV reverse transcriptase to copy genetic information from the pegRNA template to the target DNA site. The HIV RT's enhanced copying capability allows for the insertion of longer genetic sequences while maintaining high fidelity and efficiency, overcoming the limitations of MMLV RT

Inventive Principle:

Principle #26Copying

2Adaptability or versatility

If conventional prime editing methods are used, then genome editing is achieved, but flexibility and precision are limited

Engineering Contradiction:

Improveflexibility in genome editingVSAvoidediting precision

Core Design Contradiction:

Adaptability or versatilityVSManufacturing precision

Solution Approach 1:

By changing the reverse transcriptase from MMLV to HIV, the patent fundamentally improves the enzyme's ability to handle diverse genetic sequences and lengths. This parameter change enables the prime editing system to achieve both flexibility in designing various editing scenarios and precision in executing these edits, as HIV RT maintains high fidelity while accommodating longer and more diverse genetic information

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

The use of HIV reverse transcriptase-based prime editors demonstrates improved editing efficiency in human cell lines, as confirmed by phenotype and genome-level editing results, with increased CTT-to-GCC editing efficiency compared to MMLV-based systems.

Implementation Method 1

an HIV (human immunodeficiency virus) reverse transcriptase (RT) or a variant thereof

Methodology Applied

Scientific EffectReverse transcriptase: Enzyme

Implementation Method 2

a target-specific nuclease or a variant thereof

Methodology Applied

Scientific EffectNuclease: Enzyme

Data Source

PatentUS20240018492A1Prime editing using HIV reverse transcriptase and cas9 or variant thereof

Publication Date: 2024.01.18 INST FOR BASIC SCI

AI summary

The present invention relates to: a prime editing composition comprising a prime editor protein or a nucleic acid encoding same, and a prime editing guide RNA (pegRNA); and a prime editing method.