Modified sgRNA Internal Anchors for CRISPR Editing

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Solution Overview

Problem

Current gene editing technologies, particularly CRISPR-related systems, face challenges such as low HDR-mediated editing efficiency, high off-target rates, and translocation-related mutagenesis, limiting their advancement and wider application.

Innovation Solution

A system comprising a modified single-guide RNA (sgRNA) with internal anchors and a donor DNA, where the modified sgRNA includes a CRISPR RNA (crRNA) and a trans-active RNA (tracrRNA) with internal anchors located at least 5 nucleotides away from the ends, and the donor DNA has a first portion with binding segments that non-covalently bind to the internal anchors via Watson-Crick interactions, and a second portion containing a sequence of interest (SOI).

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If conventional CRISPR gene editing systems are used, then the basic gene editing function is achieved, but the HDR-mediated editing efficiency is low

Engineering Contradiction:

ImproveHDR-mediated editing efficiencyVSAvoiddesired editing outcome rate

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent introduces an intermediary binding segment that mediates between the donor DNA and the sgRNA. This binding segment, which is complementary to an internal anchor sequence in the sgRNA, acts as a bridge to enhance the interaction between the editing components, thereby improving HDR-mediated editing efficiency and desired editing outcomes.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent applies local quality by introducing specific internal anchor sequences at defined positions within the sgRNA structure. These anchors are located at least 5 nucleotides away from the 3' and 5' ends, creating localized binding sites that specifically enhance HDR efficiency without affecting other regions of the sgRNA.

Inventive Principle:

Principle #3Local quality

2Productivity

If conventional CRISPR systems are used, then gene editing is achieved, but off-target effects occur at high rates

Engineering Contradiction:

Improvegene editing rateVSAvoidoff-target effects

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The internal anchors are strategically positioned within the sgRNA structure, creating localized binding regions that enhance specificity. By concentrating the binding interaction at specific internal locations rather than the ends, the system achieves higher editing rates while reducing off-target effects through more precise targeting.

Inventive Principle:

Principle #3Local quality

3Productivity

If conventional CRISPR systems are used, then gene editing is achieved, but translocation-related mutagenesis occurs at high rates

Engineering Contradiction:

Improveediting outcome rateVSAvoidtranslocation-related mutagenesis

Core Design Contradiction:

ProductivityVSObject-generated harmful factors

Solution Approach 1:

The binding segment acts as an intermediary that facilitates controlled interactions between the donor DNA and sgRNA. This intermediary mechanism ensures that editing occurs at the intended target site through proper alignment and binding, thereby reducing translocation-related mutagenesis while maintaining high editing outcome rates.

Inventive Principle:

Principle #24Intermediary (Mediator)

4Reliability

If internal anchors are added to the sgRNA, then editing specificity and efficiency are enhanced, but the sgRNA structure becomes more complex

Engineering Contradiction:

Improveediting specificityVSAvoidsgRNA structure complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The internal anchors are introduced as localized elements within the sgRNA structure, positioned at specific locations at least 5 nucleotides away from the ends. This localized approach enhances editing specificity without requiring fundamental structural changes to the entire sgRNA, thereby minimizing the increase in overall complexity.

Inventive Principle:

Principle #3Local quality

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 system enhances the specificity and efficiency of gene editing by reducing off-target effects and translocation rates, achieving a higher percentage of desired editing outcomes compared to conventional systems.

Implementation Method 1

the first portion comprises one or more binding segments capable of binding to an internal anchor of the one or more internal anchors via a non-covalent bond

Methodology Applied

Scientific EffectWatson-Crick interaction: Chemical Bonding

Data Source

PatentUS20250177567A1Modified crispr-based gene editing system and methods of use

Publication Date: 2025.06.05 GENEDITBIO LTD

AI summary

Disclosed herein are systems comprising one or more modified single-guide RNAs (sgRNAs) and a donor DNA, wherein each of the modified sgRNAs comprises one or more internal anchors that are at least 5 nucleotides away from both 3′ and 5′ ends of each of the modified sgRNAs, wherein the donor DNA comprises one or more binding segments capable of binding to an internal anchor of the one or more internal anchors. Further disclosed herein are methods of using the systems described here