Modified Guide RNAs for CRISPR Synthesis Yield
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Solution Overview
Problem
Current gene editing tools using CRISPR/Cas systems face challenges with gRNA degradation, leading to imperfect yields and reduced stability, which affects the efficiency of gene editing, particularly in therapeutic applications.
Innovation Solution
Development of modified guide RNAs with shortened regions and substitutions, such as dual or single guide RNAs, to enhance synthesis yield, homogeneity, and stability, and improve the activity of Cas9 to cleave target DNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If standard-length guide RNAs are synthesized, then the gene editing function is maintained, but the synthesis yield is low and purity is imperfect
Solution Approach 1:
The gRNA is divided into two separate RNA molecules: crRNA containing the guide sequence and tracrRNA containing the tracr sequence. This segmentation allows each component to be synthesized independently with optimized parameters, improving overall synthesis yield and purity while maintaining the complete gRNA function when the two molecules associate.
Solution Approach 2:
The patent modifies specific parameters of the gRNA structure, including nucleotide substitutions at positions H1-1, H1-2, H1-3, H1-6 through H1-10, and other conserved regions. These parameter changes optimize the RNA structure for improved synthesis efficiency and stability while preserving Cas9 binding and DNA cleavage activity.
2Reliability
If guide RNAs are used in therapeutic applications, then gene editing is achieved, but the gRNA stability is reduced due to degradation
Solution Approach 1:
The patent creates a composite RNA system by combining crRNA and tracrRNA into a dual-guide RNA structure, or by using chemically modified single-guide RNAs with enhanced stability features. This composite approach protects the guide RNA from degradation while maintaining its ability to direct Cas9 to target DNA sequences, thereby improving reliability for therapeutic applications.
Solution Approach 2:
The patent introduces chemical modifications to the gRNA structure, including nucleotide substitutions and modifications at specific positions (H1-1, H1-2, H1-3, H1-6 through H1-10, and conserved regions). These parameter changes enhance the RNA's resistance to degradation by cellular enzymes while preserving its functional activity, extending its half-life in therapeutic contexts.
3Reliability
If the gRNA sequence is modified to improve stability, then degradation is reduced, but the synthesis complexity increases
Solution Approach 1:
By segmenting the gRNA into separate crRNA and tracrRNA molecules, the patent simplifies the synthesis process for each individual component while achieving the stability benefits of modified structures. Each segment can be synthesized using standard protocols with optimized parameters, reducing overall synthesis complexity compared to creating and optimizing a single complex modified gRNA molecule.
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 modified guide RNAs increase crude yield, purity, and homogeneity, leading to improved stability and gene editing efficiency, particularly for therapeutic applications.
Implementation Method 1
Cas9 is guided to specific DNA sequences by small RNA molecules termed guide RNA. Cas9 in combination with trRNA and crRNA or an sgRNA is termed the Cas9 ribonucleoprotein complex (RNP). Cas9 induces site-specific breaks in DNA.
Implementation Method 2
Oligonucleotides, and in particular RNA, are sometimes degraded in cells and in serum by non-enzymatic, endonuclease or exonuclease cleavage. Oligonucleotides can be synthesized with modifications at various positions to reduce or prevent such degradation.
Implementation Method 3
Cas9 is guided to specific DNA sequences by small RNA molecules termed guide RNA. The shortened regions and/or substitutions described herein may improve the activity of Cas9 (e.g., SaCas9, SpyCas9, and equivalents) to cleave target DNA.
Data Source
AI summary
This disclosure relates to modified guide RNAs having improved in vitro and in vivo activity in gene editing methods.