One-Shot Guide RNA for Transient Genome Editing
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Solution Overview
Problem
Current CRISPR/Cas-based genome editing systems face challenges with off-target effects, which pose regulatory risks for therapeutic applications.
Innovation Solution
The development of 'one-shot guide RNAs' (ogRNAs) that are adapted to temporally limit genome editing activity by engineering cellular DNA sequences recognized by gRNAs into nucleic acid sequences encoding an RNA-guided nuclease, such as Cas9 or Cpf1.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If CRISPR/Cas-based genome editing systems are used to generate precise edits at a locus of interest, then editing precision is improved, but off-target effects increase
Solution Approach 1:
The patent incorporates a governing guide RNA (ggRNA) that performs preliminary action by targeting and cleaving the Cas9 coding sequence before the Cas9 protein can cause off-target effects. This self-limiting mechanism ensures that Cas9 is activated only transiently and only at the intended target locus, preventing prolonged or off-target activity while maintaining precise editing capability.
2Productivity
If Cas9 is constitutively expressed to maintain continuous editing activity, then productivity is improved, but safety and control are worsened
Solution Approach 1:
The patent implements periodic action by designing the Cas9 expression to be transient rather than continuous. The governing guide RNA cleaves the Cas9 coding sequence after a predetermined time, creating a self-limiting expression pattern that maintains high initial editing productivity while automatically terminating the activity to ensure safety and control.
3Object-affected harmful factors
If governing guide RNA is used to limit Cas9 expression, then off-target risk is reduced, but system complexity increases
Solution Approach 1:
The patent merges the governing guide RNA function with the existing CRISPR system components. The ggRNA is integrated into the same vector or delivery mechanism as the Cas9 and target gRNA, combining multiple functions (Cas9 expression, target guiding, and self-limiting control) into a unified system that reduces overall complexity compared to separate control mechanisms.
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 reduces off-target risks by ensuring that genome editing activity is transient and specifically targeted, enhancing the predictability and safety of genome editing therapies.
Implementation Method 1
guide RNA (gRNA) which is a guide RNA targeted to the Cas9 coding sequence
Implementation Method 2
The Cas9 protein, in turn, cleaves and thereby silences the viral target
Data Source
AI summary
Engineered nucleic acids encoding genome editing system components are provided, as are engineered RNA-guided nucleases that include inserts encoded in part by cellular genomic or other sequences recognized by guide RNAs.