Chemically Modified Guide RNAs for Nuclease-Resistant CRISPR Editing

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Oligonucleotides, particularly RNA, are prone to degradation by endonucleases and exonucleases, which compromises the stability and efficiency of guide RNAs in CRISPR/Cas systems, especially in therapeutic applications.

Innovation Solution

Modified guide RNAs with specific nucleotide modifications, such as 2′-O-methyl, 2′-O-(2-methoxyethyl), 2′-fluoro, phosphorothioate bonds, and G-C substitutions, are introduced to enhance stability and activity of guide RNA/Cas9 complexes.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If guide RNA is used in CRISPR/Cas systems, then gene editing activity is achieved, but RNA degradation by endonucleases and exonucleases reduces stability and efficiency

Engineering Contradiction:

Improveguide RNA stabilityVSAvoidguide RNA half-life

Core Design Contradiction:

ReliabilityVSDuration of action of moving object

Solution Approach 1:

The patent applies parameter changes by modifying the chemical structure of nucleotides in guide RNA. Specifically, it introduces 2′-O-methyl modifications at positions 10-18 from the 5′ end and 2′-O-(2-methoxyethyl) modifications at positions 1-9 from the 5′ end. These chemical parameter changes alter the RNA's susceptibility to nucleolytic degradation, thereby extending its half-life and improving stability without compromising gene editing activity

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent employs composite materials by combining different types of modified nucleotides within the guide RNA structure. The guide RNA contains a hybrid composition of 2′-O-methyl nucleotides (providing stability) and 2′-O-(2-methoxyethyl) nucleotides (providing enhanced resistance to degradation). This composite approach creates a guide RNA that balances stability and activity better than uniform modifications

Inventive Principle:

Principle #40Composite materials

2Duration of action of moving object

If RNA modifications are introduced to improve stability, then guide RNA half-life is extended, but complexity of RNA synthesis increases

Engineering Contradiction:

Improveguide RNA half-lifeVSAvoidRNA synthesis complexity

Core Design Contradiction:

Duration of action of moving objectVSEase of manufacture

Solution Approach 1:

The patent applies local quality by implementing modifications only in specific regions of the guide RNA rather than uniformly throughout. The 2′-O-methyl modifications are placed at positions 10-18 from the 5′ end (targeting the scaffold region), while 2′-O-(2-methoxyethyl) modifications are placed at positions 1-9 from the 5′ end (targeting the 5′ overhang region). This localized modification strategy improves stability while minimizing synthesis complexity compared to full-length modifications

Inventive Principle:

Principle #3Local quality

Data Source

PatentUS20260015611A1Modified Guide RNAs

Publication Date: 2026.01.15 INTELLIA THERAPEUTICS INC

AI summary

This disclosure relates to modified single and dual guide RNAs having improved in vitro and in vivo activity in gene editing methods.