Modified Guide RNA Biotin Aptamer for Precise CRISPR Editing
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Solution Overview
Problem
Current genome editing methods using CRISPR-Cas9 systems face challenges in achieving precise editing fidelity, with imprecise insertions and deletions being more common than precise edits due to variable delivery and toxicity issues across human cell lines, and existing strategies fail to effectively control the delivery of RNPs with donor templates.
Innovation Solution
The development of modified guide RNAs, such as S1m-sgRNAs, which include a crRNA and tracrRNA hybridizing through complementary strands, incorporating an aptamer that binds a biotin-binding molecule like streptavidin, forming stable complexes with Cas9 and biotinylated donor templates to enhance precise editing through homology-directed repair.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If standard CRISPR-Cas9 delivery methods are used, then genome editing can be performed, but imprecise insertions and deletions occur more frequently than precise edits
Solution Approach 1:
The patent introduces a biotin-binding molecule as an intermediary that bridges the guide RNA and the donor template. The guide RNA is modified to include a biotin-binding aptamer, which specifically binds to biotinylated donor templates, ensuring controlled and coordinated delivery of both components to the target site, thereby reducing imprecise edits
Solution Approach 2:
The guide RNA is pre-modified with a biotin-binding aptamer sequence before delivery. This preliminary modification ensures that the guide RNA is ready to specifically capture and hold the biotinylated donor template upon arrival at the target site, preventing premature or uncontrolled interactions that lead to imprecise editing
2Productivity
If high Cas9 expression and donor template flooding are used, then editing coverage increases, but variability and toxicity across human cell lines worsen
Solution Approach 1:
The biotin-binding molecule acts as a controlled intermediary that mediates the interaction between the guide RNA and donor template. This controlled mediation allows efficient editing coverage without requiring excessive Cas9 expression or donor template flooding, thereby reducing toxicity across different human cell lines
3Manufacturing precision
If small molecules are added to block NHEJ and promote HDR, then precise editing is enhanced, but variability and toxicity across human cell lines increase
Solution Approach 1:
Instead of using small molecules to block NHEJ, the patent employs a biotin-binding intermediary to specifically recruit the donor template to the target site. This approach promotes HDR precision through controlled template delivery without the variability and toxicity associated with small molecule inhibitors across different human cell lines
4Measurement precision
If selection strategies are used to isolate precisely-edited cells, then editing fidelity improves, but imprecise editing through NHEJ still outnumbers precise HDR outcomes
Solution Approach 1:
The biotin-binding intermediary ensures that the donor template is specifically recruited and held at the target site by the modified guide RNA. This controlled recruitment increases the proportion of precise HDR outcomes relative to imprecise NHEJ events, improving both editing fidelity and the ratio of precise to imprecise edits without requiring selection strategies
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 S1mplex approach significantly increases the ratio of precise to imprecise editing, achieving up to 18-fold higher precise edits compared to standard methods, with enhanced delivery and reduced off-target effects, allowing for more controlled and efficient gene editing.
Implementation Method 1
an aptamer that binds a biotin-binding molecule
Implementation Method 2
the crRNA and the tracrRNA hybridize through the first and second complementary strands of the binding region for the Cas9 polypeptide
Implementation Method 3
forming stable complexes with Cas9
Data Source
AI summary
Described herein are modified guide RNAs such as a single guide RNA including, from 5′ to 3′, a single-stranded protospacer sequence, a first complementary strand of a binding region for the Cas9 polypeptide, an aptamer that binds a biotin-binding molecule, and a second complementary strand of the binding region for the Cas9 polypeptide. Also described is an RNP complex including the modified guide RNA and a Cas9 polypeptide or active fragment thereof. Also included are methods of modifying target genes in cells using the modified guide RNAs.