Chemically Modified sgRNA for CRISPR Delivery
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The efficient delivery of the CRISPR/Cas9 system for therapeutic applications is hindered by the need for safer and more effective delivery systems, particularly for in vitro, ex vivo, and in vivo applications, including embryo injections, due to challenges in targeting specific genomic regions and minimizing off-target effects.
Innovation Solution
Chemically modified small guide RNAs (sgRNAs) with specific modifications, such as fluorinated nucleotides, are developed to enhance binding affinity and specificity to the CRISPR/Cas system, allowing for improved targeting and reduced off-target activity, comprising a DNA-binding domain, a Cas-protein binding domain, and a transcription terminator domain with varying percentages of modified nucleotides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If chemically modified sgRNAs are used to enhance binding affinity and specificity, then genome editing efficiency is improved, but the complexity of the delivery system increases
Solution Approach 1:
The patent applies parameter changes by modifying the chemical structure of sgRNA molecules through fluorinated nucleotide substitutions and controlled phosphorothioate modifications. These chemical parameter changes enhance binding affinity and specificity to Cas9, thereby improving genome editing efficiency while managing delivery system complexity through systematic modification patterns.
Solution Approach 2:
The patent creates composite nucleic acid structures by combining modified nucleotides (fluorinated and phosphorothioate) with standard nucleotides in specific ratios and positions. This composite approach allows optimization of binding properties while maintaining deliverability, resolving the contradiction between enhanced efficiency and delivery complexity.
2Reliability
If higher percentages of modified nucleotides are used in sgRNA, then binding affinity to Cas9 is enhanced, but the risk of off-target effects increases
Solution Approach 1:
The patent applies local quality by strategically positioning modified nucleotides in specific regions of the sgRNA molecule. Fluorinated nucleotides are placed in the seed region and Cas9-binding domain where they enhance affinity, while phosphorothioate modifications are distributed in specific patterns. This localized modification strategy optimizes binding affinity while minimizing off-target effects through controlled spatial distribution of chemical modifications.
3Reliability
If chemically modified sgRNAs are developed for therapeutic applications, then safety and effectiveness are improved, but the manufacturing process complexity increases
Solution Approach 1:
The patent applies segmentation by dividing the sgRNA synthesis into distinct phases: first synthesizing oligonucleotides with fluorinated nucleotides, then incorporating phosphorothioate modifications at specific positions, and finally assembling the complete sgRNA with controlled modification ratios. This segmented manufacturing approach improves safety and effectiveness through precise control of modifications while managing process complexity through systematic stepwise synthesis.
Data Source
AI summary
The disclosure relates to compositions comprising and methods for chemical modification of single guide RNA (sgRNA), tracrRNA and/or crRNA used individually or in combination with one another or Cas system components. Compositions comprising modified ribonucleic acids have been designed with chemical modification for even higher efficiency as unmodified native strand of sgRNA. Administration of modified ribonucleic acids will allow decreased immune response when administered to a subject, increased stability, increased editing efficiency and facilitated in vivo delivery of sgRNA via various delivery platforms. The disclosure also relates to methods of decreasing off-target effect of CRISPR and a CRISPR complex.