CRISPR Guide RNA Risk Assessment for Off-Target Editing
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Solution Overview
Problem
Current genome editing technologies face significant safety issues due to off-target editing events, which can lead to unintended and potentially harmful modifications in cells, particularly in therapeutic applications like CAR-T cell therapy, necessitating a need for systems and methods to assess and mitigate these risks.
Innovation Solution
A comprehensive approach involving in silico, in vitro, and cell-based methods to identify potential off-target sites and calculate risk levels for guide nucleic acids (gNAs), using databases and assays to evaluate and rank gNAs based on hazard levels, followed by iterative testing to select suitable spacer sequences for CRISPR processes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If comprehensive testing of all guide nucleic acids is performed, then safety and reliability are improved, but time and resources are consumed
Solution Approach 1:
The patent applies preliminary action by performing in silico predictions and in vitro assays before actual genome editing experiments. The system predicts off-target effects computationally and conducts preliminary binding assays to identify high-risk guide nucleic acids early, eliminating the need to test all possible gNAs exhaustively in subsequent therapeutic applications.
Solution Approach 2:
The patent implements feedback through an iterative assessment process where results from in silico predictions and in vitro binding assays feed back into guide nucleic acid selection. The system continuously refines its risk assessment based on observed binding affinities and off-target effects, allowing for dynamic adjustment of testing strategies and prioritization of high-risk candidates for further evaluation.
2Reliability
If multiple guide nucleic acids are tested, then reliability is improved, but device complexity increases
Solution Approach 1:
The patent applies segmentation by dividing the comprehensive safety assessment into distinct modular components: in silico prediction module, in vitro binding affinity assay module, and in vivo functional validation module. Each module independently evaluates specific aspects of guide nucleic acid safety, making the overall complex assessment system manageable through systematic decomposition into standardized, reusable assessment units.
3Productivity
If binding affinity is increased for target site, then editing efficiency is improved, but off-target effects increase
Solution Approach 1:
The patent applies local quality by evaluating different regions and characteristics of guide nucleic acids individually. The system assesses binding affinity at the target site locally while simultaneously evaluating potential off-target binding sites, and uses this localized analysis to design gNAs with optimized specificity. This allows enhancement of editing efficiency at the intended target without compromising overall safety by identifying and eliminating locally problematic sequences.
Data Source
AI summary
CRISPR-Cas-based genome editing technologies demonstrate great potential as tools to facilitate gene therapy for hereditary diseases, as well as therapies that are not amenable to conventional gene therapy. However, CRISPR-Cas-based genome editing technologies may demonstrate off-target genome editing that may affect their therapeutic efficacy or other aspects. Provided herein are systems and methods to assess the hazard levels of unintended genome editing events.