CTL-seq DNA Tagging for Accurate CRISPR Off-Target Detection

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Current methods for identifying and nominating CRISPR off-target editing sites lack accuracy and sensitivity, leading to imperfect detection of mutations and changes in gene function.

Innovation Solution

A method involving co-delivery of guide RNA and unique, predefined sequence tags to cells, followed by DNA fragmentation, ligation, amplification, and sequencing to identify on- and off-target CRISPR editing sites, utilizing suppression PCR and rhAmpSeq technology to enhance specificity and sensitivity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If current methods (GUIDE-Seq, BLISS, CIRCLE-Seq) are used to identify off-target editing sites, then detection capability is provided, but accuracy and sensitivity remain imperfect

Engineering Contradiction:

Improvedetection accuracyVSAvoidsensitivity

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

The patent introduces an exogenous DNA tag sequence as an intermediary element that integrates into the genome at DSB sites. This tag serves as a mediator between the nuclease cutting event and the detection system, enabling highly sensitive and accurate detection through PCR amplification and sequencing of the tag-containing fragments, thereby resolving the contradiction between accuracy and sensitivity in off-target site identification

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent performs preliminary action by pre-designing and co-delivering unique DNA tag sequences alongside the guide RNA and nuclease. These tags are prepared in advance with specific sequences that enable selective amplification, allowing the method to achieve high sensitivity and accuracy by pre-establishing the detection framework before the actual cutting and detection events occur

Inventive Principle:

Principle #10Preliminary action

2Reliability

If cellular methods are used to identify editing targets, then actual editing targets are better identified, but comprehensive detection is limited

Engineering Contradiction:

Improveediting target identification accuracyVSAvoidcomprehensive detection coverage

Core Design Contradiction:

ReliabilityVSMeasurement precision

Solution Approach 1:

The patent applies universality by designing a detection system that works across both cellular and biochemical contexts. The DNA tag integration method and subsequent PCR/sequencing workflow can detect editing events regardless of whether they occur in living cells or in extracted DNA, providing comprehensive detection coverage while maintaining accurate identification of actual editing targets through the universal tag-based approach

Inventive Principle:

Principle #6Universality (Multi-functionality)

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

Improves the accuracy and sensitivity of detecting CRISPR off-target editing sites, reducing false positives and enhancing the reliability of identifying genetic mutations.

Implementation Method 1

CRISPR systems, such as Cas9 and Cas12a proteins, are guided to their target by RNA oligonucleotide sequences bound by the Cas proteins (forming ribonucleoprotein protein; RNP), where the enzyme creates double stranded breaks (DSBs) in DNA sequences

Methodology Applied

Scientific EffectSite-specific DNA cleavage: Enzyme