Cas12a-HUH Fusion for Targeted DNA Template Integration

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Solution Overview

Problem

Current CRISPR nucleases, such as Cas9 and Cas12a, struggle with efficient site-directed integration of template sequences into target DNA molecules, limiting their effectiveness in genetic editing.

Innovation Solution

Tethering Cas12a nucleases to HUH endonucleases using a novel linker amino acid to enhance the integration of template sequences into target DNA molecules, leveraging the HUH recognition sequence for improved cleavage and integration.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If CRISPR nucleases (Cas9, Cas12a) are used for targeted genome editing, then site-specific cleavage of DNA is achieved, but efficient site-directed integration of template sequences is limited

Engineering Contradiction:

Improveintegration efficiencyVSAvoidcleavage effectiveness

Core Design Contradiction:

Manufacturing precisionVSReliability

Solution Approach 1:

The patent combines two different nuclease systems (CRISPR Cas12a and HUH endonuclease) into a single fusion protein. The CRISPR domain provides targeted DNA binding and initial cleavage, while the HUH endonuclease domain provides additional cleavage activity and template integration capability. This merging resolves the contradiction by integrating both cleavage effectiveness and integration efficiency into one unified system.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The fusion protein creates a composite enzymatic system that combines properties of two distinct nucleases. The CRISPR Cas12a portion provides RNA-guided targeting and cleavage, while the HUH endonuclease portion provides phosphotyrosine-mediated DNA cleavage and template strand annealing. This composite structure enables both high cleavage effectiveness and improved integration efficiency simultaneously.

Inventive Principle:

Principle #40Composite materials

2Productivity

If template sequences are integrated into target DNA using standard CRISPR methods, then genome editing occurs, but integration efficiency remains low

Engineering Contradiction:

Improveintegration efficiencyVSAvoidsystem structure

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

By fusing the HUH endonuclease to the CRISPR Cas12a protein, the system combines template integration capability with targeted cleavage in a single molecular complex. This eliminates the need for separate integration enzymes or complex multi-step protocols, thereby improving integration efficiency without proportionally increasing system complexity.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The fusion protein performs multiple functions: targeted DNA binding via CRISPR, DNA cleavage via both CRISPR and HUH domains, and template integration facilitation. This multi-functionality allows a single system to handle both cleavage and integration steps, improving productivity while keeping the overall system architecture relatively simple.

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

The approach significantly enhances the efficiency of targeted integration of template sequences into DNA, achieving higher indel rates and improved integration outcomes through mechanisms like non-homologous end-joining and homologous recombination.

Implementation Method 1

HUH endonucleases are nucleases comprising a HUH (histidine-hydrophobic amino acid-histidine) tag that can form covalent bonds with specific single-stranded DNA sequences

Methodology Applied

Scientific EffectCovalent bonding: Chemical Bonding

Implementation Method 2

The HUH endonuclease is believed to function by forming a phosphotyrosine linkage to the 5' end of a nicked DNA strand

Methodology Applied

Scientific EffectPhosphotyrosine linkage: Chemical Bonding

Implementation Method 3

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) nucleases (e.g., Cas12a, CasX, Cas9) are proteins guided by guide RNAs to a target nucleic acid molecule, where the nuclease can cleave one or two strands of a target nucleic acid molecule

Methodology Applied

Scientific EffectEndonuclease cleavage: Enzyme

Implementation Method 4

achieving higher indel rates and improved integration outcomes through mechanisms like non-homologous end-joining and homologous recombination

Methodology Applied

Scientific EffectNon-homologous end-joining:

Implementation Method 5

achieving higher indel rates and improved integration outcomes through mechanisms like non-homologous end-joining and homologous recombination

Methodology Applied

Scientific EffectHomologous recombination:

Data Source

PatentUS20250382624A1Methods and compositions to promote targeted genome modifications using HUH endonucleases

Publication Date: 2025.12.18 MONSANTO TECHNOLOGY LLC

AI summary

This disclosure provides compositions and methods for improving site-directed integration of nucleic acids using RNA-guided nucleases coupled with HUH endonucleases.