Class 2 Type V Cas12a Prime Editing With Nickase Activity

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Solution Overview

Problem

Class 2 type V CRISPR-Cas systems lack specific nickases, such as Cas9 H840A nickase, hindering precise genome editing, and existing systems like REDRAW suffer from off-target effects and inefficiencies.

Innovation Solution

A prime editing system utilizing a Cas12a enzyme with nickase activity, linked to a reverse transcriptase through a linker, and a pegRNA system for precise editing without double-strand breaks, including an organellar localization signal and ssDNA-binding proteins.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If class 2 type V CRISPR-Cas systems are used for prime editing, then the system can perform genome editing, but specific nickases are not available which hinders precise editing

Engineering Contradiction:

Improveprime editing capabilityVSAvoidprecise editing

Core Design Contradiction:

Adaptability or versatilityVSEase of operation

Solution Approach 1:

The patent divides the Cas12a enzyme into functional domains: the RuvC domain is used for nickase activity on the non-target strand, while the Nuc domain coordinates substrate DNA. This segmentation allows independent optimization of nicking function without requiring a completely new enzyme design.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent makes Cas12a multi-functional by combining nickase activity (via RuvC domain) with reverse transcriptase activity, allowing a single enzyme system to perform both strand nicking and template synthesis without requiring separate specialized enzymes.

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Productivity

If Cas12a cleaves both DNA strands sequentially using a single catalytic site, then the system can perform editing, but this structural organization hampers the design of true nickases

Engineering Contradiction:

Improveediting capabilityVSAvoidnickase design

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent applies local quality by making the RuvC domain specifically active for non-target strand nicking while the Nuc domain handles substrate coordination. This localized functional assignment creates a specialized nicking mechanism within the broader Cas12a system.

Inventive Principle:

Principle #3Local quality

3Productivity

If a nuclease is used in the editing system, then editing can be performed, but off-target effects and undesired indels increase which reduces precise editing efficiency

Engineering Contradiction:

Improveediting efficiencyVSAvoidprecise editing

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent converts the potential harm of non-specific nuclease activity into a benefit by using the RuvC domain's controlled nicking activity specifically for non-target strand cleavage. This targeted approach prevents unwanted indels while maintaining editing efficiency.

Inventive Principle:

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

Solution Approach 2:

The patent changes the catalytic parameter of Cas12a from double-strand breaking to single-strand nicking by utilizing the RuvC domain's specific activity, thereby reducing off-target effects and improving editing precision.

Inventive Principle:

Principle #35Parameter changes

4Adaptability or versatility

If classical prime editors based on Cas9 are used, then prime editing can be performed, but the system cannot be extended to modern class 2 type V CRISPR-Cas systems

Engineering Contradiction:

Improvesystem compatibilityVSAvoidediting precision

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The patent creates a universal prime editing platform by adapting the Cas12a enzyme to perform multiple functions: non-target strand nicking via RuvC domain, substrate coordination via Nuc domain, and integration with reverse transcriptase for template synthesis. This multi-functionality enables extension to class 2 type V systems while maintaining precision.

Inventive Principle:

Principle #6Universality (Multi-functionality)

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

Enables efficient and precise genome editing with reduced off-target effects, extending prime editing capabilities to modern Class 2 type V CRISPR-Cas systems.

Implementation Method 1

Subsequently, the sequence of the RTT sequence is reverse transcribed from the pegRNA into an edited target DNA sequence

Methodology Applied

Scientific EffectReverse transcription:

Implementation Method 2

the Cas12a enzyme, preferably having nickase activity (nCas12a), more preferably having non-target strand (NTS) nickase activity

Methodology Applied

Scientific EffectNickase activity: Enzyme

Implementation Method 3

The PBS region is complementary to the non-target strand and after base pairing, the non-target strand will create a primer

Methodology Applied

Scientific EffectBase pairing:

Data Source

PatentUS20250243482A1Class 2 type v crispr-cas prime editing

Publication Date: 2025.07.31 BASF AGRICULTURAL SOLUTIONS SEED US LLC

AI summary

The present disclosure relates to the field of gene genome editing. In particular, it relates to the provision of a CRISPR-Cas class 2 type V prime editing system, including a prime editor, a prime editor complex, a prime editing guide RNA system as well as the means and methods for the modification of a nucleic acid sequence of interest with the prime editing system of the present invention.