Guide RNA Internal Linkers for Synthesis Yield and Stability

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Current gene editing tools using CRISPR/Cas systems face challenges in the cyclic nature and imperfect yield of oligonucleotide synthesis, leading to instability and inefficiency of guide RNAs, which affects their therapeutic applications.

Innovation Solution

Incorporating non-nucleic acid internal linkers into guide RNAs to replace non-essential contacts, improving yield, homogeneity, and stability, and enhancing the activity of Cas nuclease complexes like Cas9 and Cas12.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If guide RNA is synthesized using conventional oligonucleotide synthesis, then the guide RNA can be produced, but the yield is limited and homogeneity is poor due to cyclic synthesis imperfections

Engineering Contradiction:

Improveguide RNA yieldVSAvoidguide RNA homogeneity

Core Design Contradiction:

ProductivityVSManufacturing precision

Solution Approach 1:

The guide RNA is divided into multiple segments that are chemically synthesized with high precision, then assembled together. This segmentation allows each segment to be optimized for synthesis yield and purity, while the overall guide RNA functionality is maintained through proper assembly of the segments.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The guide RNA incorporates non-nucleic acid internal linkers (such as peptide nucleic acid or other synthetic linkers) that replace traditional phosphodiester bonds in specific regions. These composite structures combine the advantages of nucleic acid sequences with the superior synthetic properties of alternative chemistries, improving both yield and homogeneity.

Inventive Principle:

Principle #40Composite materials

2Reliability

If guide RNA is used for gene editing applications, then gene editing function is achieved, but stability of the guide RNA is insufficient leading to reduced efficiency

Engineering Contradiction:

Improveguide RNA stabilityVSAvoidgene editing efficiency

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The chemical parameters of the guide RNA are modified by incorporating non-nucleic acid linkers that alter the molecular properties. These parameter changes include improved resistance to nucleases, enhanced thermal stability, and modified binding characteristics, all of which contribute to greater stability without compromising gene editing efficiency.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

Non-nucleic acid internal linkers serve as intermediary structures within the guide RNA molecule. These intermediaries provide structural support and stability while maintaining the necessary RNA-DNA hybrid formation capabilities, acting as bridges that connect functional regions of the guide RNA with improved chemical properties.

Inventive Principle:

Principle #24Intermediary (Mediator)

Data Source

PatentUS20240150761A1Modified Guide RNAs Comprising an Internal Linker for Gene Editing

Publication Date: 2024.05.09 INTELLIA THERAPEUTICS INC

AI summary

This disclosure relates to modified guide RNAs comprising an internal linker for in vitro and in vivo gene editing methods.