Cis-blocked guide RNA for CRISPR specificity
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Solution Overview
Problem
Current RNA-guided genome editing technologies, such as the CRISPR-Cas system, face challenges in specificity and efficiency due to off-target DNA cleavage, with existing strategies either reducing on-target activity or requiring additional DNA sequences for improved specificity.
Innovation Solution
The use of engineered guide RNAs with a blocking sequence that hybridizes with a portion of the guide sequence, forming a cis-blocked stem, which competes with off-target sites for binding and enhances the thermodynamic favorability of on-target binding, thereby reducing off-target cleavage while maintaining or increasing on-target activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the CRISPR-Cas9 system uses a 20-nt guide sequence with full complementarity, then on-target cleavage activity is high, but off-target DNA cleavage occurs due to tolerance of base-pair mismatching
Solution Approach 1:
The guide RNA is segmented into two functional regions: a 20-nt guide sequence for target recognition and a blocking sequence (5-15 nt) that hybridizes to a portion of the guide sequence. This segmentation allows the guide sequence to maintain its target-binding function while the blocking sequence actively prevents off-target binding by competing for hybridization, thereby resolving the contradiction between high on-target activity and reduced off-target cleavage
Solution Approach 2:
The blocking sequence performs preliminary anti-action by pre-hybridizing to the guide sequence in the absence of target DNA, creating a thermodynamically stable blocked state. This preliminary blocking prevents the guide RNA from binding to off-target sites with partial complementarity, while still allowing on-target binding to occur under appropriate conditions, thus preventing harmful off-target cleavage before it can happen
2Reliability
If existing strategies are used to improve specificity (Cas9 nickase, dCas9-FokI, truncated guide RNA), then off-target cleavage is reduced, but on-target cleavage activity is diminished
Solution Approach 1:
The invention changes the thermodynamic parameters of guide RNA binding by introducing a blocking sequence with specific length (5-15 nt) and complementarity to the guide sequence. This parameter change creates a temperature-dependent or condition-dependent equilibrium where the blocked state predominates under normal conditions (preventing off-target binding) but can be overcome by high-affinity on-target binding, thereby maintaining productivity while improving specificity
Solution Approach 2:
The blocking sequence acts as an intermediary element that mediates between the guide sequence and off-target DNA sites. By introducing this intermediate blocking layer, the system allows selective passage: on-target binding can overcome the block due to perfect complementarity and higher affinity, while off-target binding is blocked by the thermodynamically stable blocking sequence, thus resolving the contradiction between specificity and efficiency
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
This approach significantly reduces off-target base-pairing and increases the specificity of CRISPR-Cas systems, minimizing reductions in on-target cleavage activity and enhancing the stability and applicability of the guide RNA, allowing for more precise genome editing.
Implementation Method 1
a blocking sequence that hybridizes with a portion of the guide sequence
Implementation Method 2
enhances the thermodynamic favorability of on-target binding, thereby reducing off-target cleavage
Data Source
AI summary
This invention discloses reagents and methods for increasing specificity and efficiency of RNA-guided genome editing.