Prime Editing Efficiency via Cis-Acting Regulatory Elements

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Solution Overview

Problem

Current prime editing technologies face challenges in achieving high efficiency due to the large size of the Cas9(H840A) nickase-reverse transcriptase fusion protein, which hinders stable expression and efficient DNA editing, and are prone to off-target effects and undesired outcomes such as translocations and pathological alleles.

Innovation Solution

Incorporation of cis-acting regulatory elements like dENE or sRSM1 into the Cas9(H840A) nickase-reverse transcriptase expression cassette to enhance mRNA stability and protein expression, improving the efficiency of prime editing without altering the desired edits or adding extra components to the prime editing complex.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If the Cas9(H840A) nickase-reverse transcriptase fusion protein is used for prime editing, then the ability to perform targeted insertions, deletions and base-to-base conversions is achieved, but the large size of the fusion protein hinders stable expression and reduces editing efficiency

Engineering Contradiction:

Improvegenome editing capabilityVSAvoidediting efficiency

Core Design Contradiction:

Adaptability or versatilityVSProductivity

Solution Approach 1:

The patent divides the large Cas9(H840A) nickase-reverse transcriptase fusion protein into separate components: the Cas9(H840A) nickase and the reverse transcriptase. By separating these functions and delivering them independently (e.g., Cas9 nickase as ribonucleoprotein and reverse transcriptase as mRNA), the system overcomes the expression hurdles associated with the large fusion protein while maintaining the prime editing capability.

Inventive Principle:

Principle #1Segmentation

2Adaptability or versatility

If CRISPR/Cas9 systems make double-strand breaks for DNA manipulation, then insertion, deletion or base substitution can be achieved, but off-target cleavages and translocations occur

Engineering Contradiction:

ImproveDNA manipulation capabilityVSAvoidoff-target effects

Core Design Contradiction:

Adaptability or versatilityVSObject-affected harmful factors

Solution Approach 1:

The patent converts the potential harm of double-strand breaks into a beneficial prime editing mechanism. Instead of creating DSBs that cause off-target effects and translocations, the system uses a nickase to create a single-strand nick, then employs reverse transcription from an integrated reverse transcriptase to synthesize the edited sequence directly on the nicked strand, avoiding the harmful effects of DSBs while achieving precise editing.

Inventive Principle:

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

3Adaptability or versatility

If current prime editing technology is used, then targeted genome editing can be performed, but the efficiency is insufficient for research and therapeutic applications

Engineering Contradiction:

Improveprime editing functionVSAvoidediting efficiency

Core Design Contradiction:

Adaptability or versatilityVSProductivity

Solution Approach 1:

The patent introduces an integrated reverse transcriptase as an intermediary component within the prime editing complex. This reverse transcriptase uses the pegRNA template to synthesize the edited DNA sequence directly at the nick site, serving as a mediator that bridges the gap between the nickase activity and the desired editing outcome, thereby significantly improving editing efficiency.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent changes key parameters of the prime editing system by using Cas9(H840A) nickase instead of wild-type Cas9, and by integrating a reverse transcriptase with specific properties (e.g., M-MLV RT or engineered variants). These parameter changes optimize the editing efficiency while maintaining specificity and reducing off-target effects.

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 integration of cis-acting regulatory elements significantly enhances the efficiency of prime editing, stabilizing edited DNA and reducing off-target effects, thereby improving the precision and reliability of genome editing.

Implementation Method 1

The resulting 3′ end hybridizes to the primer-binding site

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

then primes reverse transcription of new DNA containing the desired edit using the transcriptase template of the pegRNA

Methodology Applied

Scientific EffectReverse transcription:

Implementation Method 3

Incorporation of cis-acting regulatory elements like dENE or sRSM1 into the Cas9(H840A) nickase-reverse transcriptase expression cassette to enhance mRNA stability and protein expression

Methodology Applied

Scientific EffectmRNA stabilization:

Implementation Method 4

Equilibration between the edited 3′ flap and the unedited 5′ flap, cellular 5′ flap cleavage and ligation, and DNA repair results in stably edited DNA

Methodology Applied

Scientific EffectDNA ligation:

Data Source

PatentUS20240409907A1Improved prime editing system efficiency with cis-acting regulatory elements

Publication Date: 2024.12.12 EMD MILLIPORE CORP

AI summary

The present invention is a synthetic nucleic acid composition comprising: i) a sequence encoding a CRISPR-Cas protein, ii) a sequence encoding a reverse transcriptase, and iii) a sequence encoding a cis-acting regulatory element, and methods of use thereof.