Modified gRNA Inverted Repeat Stabilizes Cas Protein Binding
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Solution Overview
Problem
The mechanism governing the removal of CRISPR-associated (Cas) proteins by motor proteins is not well understood, particularly in the context of CRISPR interference, where the effectiveness of Cas protein removal depends on its orientation relative to transcription, and there is a need to elucidate how Cas proteins interact with DNA to influence their binding stability.
Innovation Solution
Modified guide RNAs (gRNAs) with an inverted repeat sequence are used, which can concurrently hybridize to the spacer sequence and its complementary strand, stabilizing the R-loop and enhancing Cas protein resistance to removal by motor proteins, and expression vectors encoding these gRNAs and Cas proteins are introduced into cells to form a complex with the Cas protein and DNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If standard guide RNA is used, then Cas protein can bind to target DNA, but Cas protein is easily removed by motor proteins
Solution Approach 1:
The patent merges the guide RNA with an inverted repeat sequence that can form a hairpin structure, creating a unified molecular entity that simultaneously guides Cas protein binding and stabilizes the complex through secondary structure formation. This combined structure resists motor protein removal more effectively than standard guide RNA alone.
Solution Approach 2:
The inverted repeat sequence is designed to pre-form a stable hairpin structure before Cas protein binding occurs. This preliminary structural organization creates a more stable R-loop complex that is resistant to subsequent motor protein action, preventing easy removal of the Cas protein from the DNA target.
2Reliability
If guide RNA binding stability is increased, then Cas protein resistance to removal improves, but R-loop formation complexity increases
Solution Approach 1:
The guide RNA contains an inverted repeat sequence that automatically forms a hairpin structure through intramolecular base pairing. This self-organizing property eliminates the need for external factors or complex assembly mechanisms, as the RNA molecule autonomously creates its stabilizing secondary structure upon binding to the Cas protein and target DNA.
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 gRNAs improve Cas protein resistance to removal by motor proteins, modulating its function and stability, thereby enhancing the efficacy of CRISPR interference and gene editing processes.
Implementation Method 1
hybridization of the spacer region of the gRNA with the target DNA to form a gRNA/DNA hybrid (R-loop)
Implementation Method 2
The inverted repeat sequence is configured so that it can concurrently be hybridized to the spacer sequence and to the complementary strand of the DNA comprising the spacer sequence
Data Source
AI summary
Provided are modified guide RNAs (gRNAs) for use with CRISPR Cas proteins. A modified guide RNA comprises at its 5′ or 3′end at least 5 nucleotides that comprise an inverted repeat sequence having a segment targeted to a spacer sequence in DNA. The inverted repeat sequence is configured so that it can concurrently be hybridized to the spacer sequence and to the complementary strand of the DNA comprising the spacer sequence when in the presence of the DNA and the Cas protein. The modified gRNA influences the Cas protein interaction with DNA.