CRISPR-Cas9 Plasmid Linearization for Homogeneous Poly(A) RNA

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Solution Overview

Problem

Current methods for producing linear DNA templates for RNA synthesis, such as using type II restriction enzymes or Rho-independent terminators, result in inefficient, costly, and complex processes with unwanted transcript elongations or poly(A) attachment inhomogeneities, limiting the scalability and efficiency of RNA production.

Innovation Solution

A CRISPR-Cas system is employed to linearize circular DNA molecules by using a guide RNA and Cas nuclease, specifically targeting a protospacer adjacent motif (PAM) to create smooth ends with a poly T sequence, enabling homogeneous poly(A) attachment for RNA synthesis.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If type II restriction enzymes are used to linearize plasmid DNA, then the DNA can be cut at defined positions, but the process becomes complex and produces unwanted overhangs requiring additional processing steps

Engineering Contradiction:

Improveprecision of DNA cuttingVSAvoidcomplexity of linearization process

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The patent extracts only the essential function of restriction enzymes (cutting DNA at specific positions) while removing the problematic features (overhang production). CRISPR-Cas9 is used to achieve precise cutting without generating overhangs, eliminating the need for additional processing steps to remove unwanted ends.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent employs a disposable CRISPR guide RNA designed to direct Cas9 to the exact cutting position. This single-use guide RNA system replaces the need for complex, multi-step restriction enzyme protocols and subsequent cleanup procedures, simplifying the overall process.

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

2Productivity

If type IIS restriction enzymes are used to generate linear DNA fragments, then the DNA is linearized efficiently, but 3' overhangs are produced that cause promiscuous transcription and unwanted elongated RNAs

Engineering Contradiction:

Improveefficiency of DNA linearizationVSAvoidunwanted transcript elongation

Core Design Contradiction:

ProductivityVSObject-generated harmful factors

Solution Approach 1:

The patent converts the potential harm of overhang-induced promiscuous transcription into a benefit by using CRISPR-Cas9 to create clean, precise cuts without overhangs. This eliminates the harmful effect while maintaining the efficiency of linearization, producing only the desired full-length transcripts without unwanted elongations.

Inventive Principle:

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

3Manufacturing precision

If Rho-independent terminators are used to control transcript length, then RNA synthesis is halted at defined positions, but the process requires additional steps to remove terminator sequences and reduces overall efficiency

Engineering Contradiction:

Improvecontrol of transcript lengthVSAvoidefficiency of RNA production

Core Design Contradiction:

Manufacturing precisionVSProductivity

Solution Approach 1:

The patent extracts the essential function of Rho-independent terminators (controlling transcript length) while removing the cumbersome aspects (additional removal steps). CRISPR-Cas9 linearization provides precise control of the DNA template end position, achieving the same effect without requiring subsequent terminator sequence removal.

Inventive Principle:

Principle #2Taking out (Extraction)

4Stability of the object's composition

If poly(A) polymerase is used to add poly(A) attachments to RNA, then the RNA stability is improved, but the poly(A) attachment length becomes inhomogeneous requiring additional purification steps

Engineering Contradiction:

Improvestability of RNAVSAvoidhomogeneity of poly(A) attachment

Core Design Contradiction:

Stability of the object's compositionVSManufacturing precision

Solution Approach 1:

The patent performs preliminary action by incorporating the poly(A) attachment sequence directly into the DNA template during the linearization step. CRISPR-Cas9 is used to create a DNA template with a defined poly(T) sequence at the 3' end, which serves as the template for synthesizing homogeneous poly(A) attachments during in vitro transcription, eliminating the need for subsequent poly(A) polymerase treatment and purification.

Inventive Principle:

Principle #10Preliminary action

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 method simplifies the DNA linearization process, reduces costs, and enhances the homogeneity and efficiency of RNA production, particularly for mRNA synthesis, improving stability and translation efficiency.

Implementation Method 1

A CRISPR-Cas system is employed to linearize circular DNA molecules by using a guide RNA and Cas nuclease, specifically targeting a protospacer adjacent motif (PAM) to create smooth ends

Methodology Applied

Scientific EffectCRISPR-Cas9 genome editing: Enzyme

Data Source

PatentEP4544037B1Crispr-cas9-based linearization of plasmid DNA templates

Publication Date: 2026.01.21 WACKER CHEMIE AG

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AI summary

The invention relates to a method for producing a linear double-stranded DNA molecule, wherein the linear DNA molecule has smooth ends, and wherein the linear DNA molecule has a poly T sequence at the 5' end of the matrix strand, using a leader DNA and a Cas nuclease.