Chemically Modified Guide RNAs for High Specificity Genome Editing

Solution Overview

Problem

Current CRISPR-Cas systems face challenges in achieving specificity, leading to off-target effects, which compromise the efficacy of gene editing by causing unintended cleavage at non-target sites.

Innovation Solution

Chemical modifications to guide RNAs (gRNAs) are introduced to weaken or strengthen nucleotide pair associations, specifically in the guide sequence, locking region, sampling region, and seed region, to enhance specificity by altering melting temperatures and binding energies, thereby reducing off-target interactions while maintaining on-target efficacy.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If chemical modifications are introduced to guide RNA to enhance specificity, then off-target effects are reduced, but the complexity of guide RNA synthesis increases

Engineering Contradiction:

Improvespecificity of guide RNAVSAvoidcomplexity of guide RNA synthesis

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The patent applies parameter changes by introducing chemical modifications to the guide RNA structure, specifically altering the sugar moiety with 2'-O-methyl groups and modifying the phosphate backbone with phosphorothioate linkages. These parameter changes in the chemical structure enhance the melting temperature and binding stability of the guide RNA, thereby reducing off-target effects while maintaining on-target specificity.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent employs composite materials by combining multiple chemical modifications within the guide RNA molecule. The guide RNA consists of a hybrid structure incorporating both 2'-O-methyl modified nucleotides and phosphorothioate internucleotide linkages, creating a composite nucleic acid structure that leverages the beneficial properties of each modification type to achieve enhanced specificity and stability.

Inventive Principle:

Principle #40Composite materials

2Manufacturing precision

If chemical modifications are introduced to guide RNA to reduce off-target effects, then specificity is improved, but the cost and difficulty of manufacture increase

Engineering Contradiction:

Improvespecificity of guide RNAVSAvoidease of guide RNA synthesis

Core Design Contradiction:

Manufacturing precisionVSEase of manufacture

Solution Approach 1:

The patent applies parameter changes by introducing chemical modifications to the guide RNA structure, specifically altering the sugar moiety with 2'-O-methyl groups and modifying the phosphate backbone with phosphorothioate linkages. These parameter changes in the chemical structure enhance the melting temperature and binding stability of the guide RNA, thereby reducing off-target effects while maintaining on-target specificity.

Inventive Principle:

Principle #35Parameter changes

3Manufacturing precision

If chemical modifications are introduced to guide RNA to alter melting temperatures, then binding specificity is enhanced, but the functional activity may be compromised

Engineering Contradiction:

Improvebinding specificityVSAvoidfunctional activity of guide RNA

Core Design Contradiction:

Manufacturing precisionVSReliability

Solution Approach 1:

The patent applies parameter changes by introducing chemical modifications to the guide RNA structure, specifically altering the sugar moiety with 2'-O-methyl groups and modifying the phosphate backbone with phosphorothioate linkages. These parameter changes in the chemical structure enhance the melting temperature and binding stability of the guide RNA, thereby reducing off-target effects while maintaining on-target specificity.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent applies local quality by strategically positioning chemical modifications at specific regions of the guide RNA molecule. The modifications are concentrated in the seed region (nucleotides 10-20) and offset region (nucleotides 1-9), with different types and densities of modifications applied to different regions based on their functional requirements, thereby optimizing both specificity and activity locally.

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

The chemical modifications significantly decrease off-target effects, as evidenced by higher specificity scores and reduced cleavage of non-target polynucleotides, while preserving the ability to effectively bind, nick, or cleave target polynucleotides, thus improving the precision of CRISPR-Cas systems.

Implementation Method 1

enhance specificity by altering melting temperatures and binding energies

Methodology Applied

Scientific EffectMelting temperature:

Implementation Method 2

enhance specificity by altering melting temperatures and binding energies

Methodology Applied

Scientific EffectBinding energy:

Data Source

PatentUS20200339980A1High Specificity Genome Editing Using Chemically Modified Guide RNAs

Publication Date: 2020.10.29 AGILENT TECHNOLOGIES INC

AI summary

The present invention relates to guide RNAs having chemical modifications and their use in CRISPR-Cas systems. The chemically modified guide RNAs have enhanced specificity for target polynucleotide sequences. The present invention also relates to methods of using chemically modified guide RNAs for cleaving or nicking polynucleotides, and for high specificity genome editing.