Chemically Modified Nme Guide RNAs for Stable Genome Editing

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Solution Overview

Problem

Existing guide RNAs for CRISPR genome editing, such as those guided by Cas9, face limitations including rapid degradation and reduced stability, which affects their efficiency and therapeutic applications, and they can induce immunogenic responses.

Innovation Solution

Development of heavily or fully chemically modified Neisseria meningitidis (Nme) guide RNAs with specific modifications in the crRNA and tracrRNA portions, including 2′-O-methyl, 2′-fluoro, and 2′-deoxy modifications, to enhance stability, potency, and reduce off-target effects and immunogenicity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Stability of the object's composition

If existing guide RNAs are used for CRISPR genome editing, then genome editing function is achieved, but rapid degradation occurs and stability is reduced

Engineering Contradiction:

Improveguide RNA stabilityVSAvoidguide RNA circulation time

Core Design Contradiction:

Stability of the object's compositionVSDuration of action of moving object

Solution Approach 1:

The patent applies chemical modifications to the guide RNA structure, specifically incorporating 2'-O-methyl modifications at certain positions and 2'-fluoro modifications at other positions. These chemical parameter changes enhance the stability of the guide RNA against degradation while maintaining its ability to guide Cas9 for genome editing, thereby extending its duration of action in circulation and within cells.

Inventive Principle:

Principle #35Parameter changes

2Object-affected harmful factors

If existing guide RNAs are used for CRISPR genome editing, then editing function is achieved, but immunogenic responses are induced

Engineering Contradiction:

ImproveimmunogenicityVSAvoidtherapeutic applicability

Core Design Contradiction:

Object-affected harmful factorsVSReliability

Solution Approach 1:

The patent modifies the guide RNA chemical structure by incorporating 2'-O-methyl and 2'-fluoro modifications, which alter the immunogenic properties of the RNA. These parameter changes reduce the ability of the guide RNA to trigger innate immune responses while preserving its genome editing functionality, thereby improving therapeutic applicability and reliability.

Inventive Principle:

Principle #35Parameter changes

3Stability of the object's composition

If chemical modifications are added to guide RNAs to improve stability, then stability increases, but editing efficiency may be reduced

Engineering Contradiction:

Improveguide RNA stabilityVSAvoidediting efficiency

Core Design Contradiction:

Stability of the object's compositionVSManufacturing precision

Solution Approach 1:

The patent applies a localized modification strategy where 2'-O-methyl modifications are placed at specific positions (such as positions 1-10 and 20-30) and 2'-fluoro modifications at other specific positions (such as positions 11-19 and 21-29) of the guide RNA sequence. This local quality approach ensures that stability-enhancing modifications are concentrated in regions where they provide maximum protection without interfering with the critical seed region and PAM recognition elements, thereby maintaining high editing efficiency while achieving improved stability.

Inventive Principle:

Principle #3Local quality

Data Source

PatentUS20230183687A1Modified guide rnas for neisseria meningitidis cas9

Publication Date: 2023.06.15 UNIV OF MASSACHUSETTS

AI summary

Chemically modified Neisseria meningitidis (Nme) crRNAs, tracrRNAs, and sgRNAs are provided. Methods of using the Nme crRNAs, tracrRNAs, and sgRNAs for genome editing with a Nme CRISPR nuclease and kits for performing the same are also provided.