NHEJ-Mediated DNA Sequence Replacement

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Solution Overview

Problem

Current methods for modifying double-stranded DNA using RNA-guided nucleases, such as CRISPR/Cas9, face inefficiencies in replacing DNA sequences, particularly in non-dividing cells and mammalian systems, with limitations in precision and flexibility due to reliance on homologous recombination and the generation of unwanted insertions and deletions.

Innovation Solution

An in vitro method employing an RNA-guided DNA endonuclease to generate two double-strand breaks in a dsDNA molecule, allowing for targeted replacement of the sequence between these breaks using the non-homologous end joining (NHEJ) pathway, without the need for homology arms or specific single-strand overhangs, enabling precise and controlled substitution of DNA sequences.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If homologous recombination or single strand template repair (SSTR) is used to replace DNA sequences, then precise sequence replacement can be achieved, but the efficiency is low and the method is restricted to dividing cells in S/G2 phases

Engineering Contradiction:

Improveprecision of sequence replacementVSAvoidefficiency of sequence replacement

Core Design Contradiction:

Manufacturing precisionVSProductivity

Solution Approach 1:

The invention changes the fundamental parameter of the repair pathway from homologous recombination/SSTR to non-homologous end joining (NHEJ). This parameter change enables the repair process to occur in all cell cycle phases (including G0/G1 where non-dividing cells reside) rather than being restricted to S/G2 phases, thereby dramatically increasing productivity while maintaining acceptable precision through the use of designed donor sequences

Inventive Principle:

Principle #35Parameter changes

2Manufacturing precision

If homologous recombination or SSTR is used for sequence replacement, then precise editing can be achieved in theory, but the method is restricted to dividing and cycling cells only

Engineering Contradiction:

Improveprecision of DNA editingVSAvoidapplicability to different cell types

Core Design Contradiction:

Manufacturing precisionVSAdaptability or versatility

Solution Approach 1:

The invention fundamentally changes the cell cycle phase parameter by utilizing NHEJ pathway instead of homologous recombination. NHEJ is active throughout all cell cycle phases including G0/G1, making the method applicable to non-dividing cells such as neurons, muscle cells, and differentiated mammalian cells, thereby dramatically expanding adaptability and versatility across different cell types

Inventive Principle:

Principle #35Parameter changes

3Measurement precision

If RNA-guided nucleases are used to generate double strand breaks for sequence replacement, then targeted editing capability is improved, but unwanted insertions and deletions (INDELs) are generated at the cut sites

Engineering Contradiction:

Improvetargeting precision of DNA editingVSAvoidunwanted insertions and deletions

Core Design Contradiction:

Measurement precisionVSObject-generated harmful factors

Solution Approach 1:

The invention extracts and removes the problematic INDEL formation by using donor DNA sequences that are specifically designed to be ligated into the cut sites. The donor sequences contain the desired replacement payload flanked by sequences complementary to the ends generated by the RNA-guided nuclease, allowing precise integration without the random INDELs that would otherwise occur during NHEJ repair

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invention introduces donor DNA sequences as an intermediary element that mediates the repair process. These donor sequences serve as templates that guide the NHEJ pathway to insert the desired payload precisely at the cut sites, acting as a bridge between the nuclease-induced breaks and the desired edited outcome, thereby preventing unwanted INDEL formation

Inventive Principle:

Principle #24Intermediary (Mediator)

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 achieves high-frequency, precise, and controlled DNA sequence replacement without generating insertions or deletions, facilitating targeted genome editing in both dividing and non-dividing cells, including mammalian cells, with increased flexibility in choosing target sites and reduced complexity in enzyme design.

Implementation Method 1

replacing a DNA sequence of the dsDNA molecule to be modified, wherein the replaced sequence is located between the double strand breaks (between target sequences 1 and 2) and is replaced by the DNA substitute sequence of the exogenous nucleic acid molecule by the non-homologous end joining (NHEJ) pathway

Methodology Applied

Scientific EffectNon-homologous end joining (NHEJ):

Data Source

PatentUS11891635B2Nucleic acid sequence replacement by NHEJ

Publication Date: 2024.02.06 MAX DELBRUECK CENT FUER MOLEKULARE MEDIZIN

AI summary

A method for modifying double stranded DNA (dsDNA) employing an RNA guided DNA endonuclease to generate two double strand breaks in the dsDNA molecule to be modified, and replacement of the sequence positioned between the double strand breaks with a substitute DNA sequence using the non-homologous end joining (NHEJ) pathway, and corresponding kits and compositions for modifying double stranded DNA molecules.