Primary T-Cell GUIDE-Seq for Sensitive Off-Target Detection
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Solution Overview
Problem
Existing methods for identifying off-target genome editing sites in primary human cells, such as GUIDE-Seq, are limited by the cytotoxicity of double-stranded oligonucleotides and cannot account for patient-specific genomic variants, leading to unreliable detection of off-target effects.
Innovation Solution
A method involving the use of non-viral delivery of ribonucleoproteins (RNPs) complexed with TRAC or TRBC guide RNAs and a double-stranded oligonucleotide (dsODN) in primary human T cells, followed by unbiased amplification and sequencing to identify off-target effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If GUIDE-Seq method using double-stranded oligonucleotides is applied to primary human T cells, then off-target detection sensitivity is improved, but cell cytotoxicity increases and reliability deteriorates
Solution Approach 1:
The patent introduces a novel intermediary molecule: a single-stranded oligonucleotide with a 5' phosphate group that serves as a marker for double-strand breaks. This intermediary integrates into the DNA break sites and enables detection without requiring the cytotoxic double-stranded oligonucleotides used in traditional GUIDE-Seq, thus resolving the contradiction between detection sensitivity and cell cytotoxicity
Solution Approach 2:
The patent changes the fundamental parameter of the oligonucleotide marker from double-stranded to single-stranded configuration. This parameter change eliminates the cytotoxicity issue while maintaining the ability to mark and detect double-strand break sites through subsequent PCR amplification and sequencing
2Manufacturing precision
If computational simulations are used to identify off-target sites, then manufacturing precision is improved, but measurement precision deteriorates due to inability to detect in vitro off-target sites
Solution Approach 1:
The patent creates a molecular copy of the DNA break site through integration of the single-stranded oligonucleotide marker. This copy serves as a detectable proxy for the actual break site, enabling direct experimental measurement of off-target effects in vitro rather than relying on computational predictions, thus resolving the contradiction between prediction accuracy and measurement reliability
3Measurement precision
If existing GUIDE-Seq method is used, then off-target detection is improved, but adaptability deteriorates due to inability to account for patient-specific genomic variants
Solution Approach 1:
The patent develops a universal detection system based on single-stranded oligonucleotide integration that works across different cell types and patient samples. The method universally detects double-strand breaks regardless of patient-specific genomic variants, enabling adaptable off-target screening in primary human cells with diverse genetic backgrounds
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
Enables highly sensitive and accurate detection of off-target genome editing sites in primary human T cells, overcoming limitations of existing methods by providing patient-specific and unbiased identification of off-target cleavage.
Implementation Method 1
providing a nuclease composition capable of inducing a double-stranded break in the genomic DNA of a primary cell
Implementation Method 2
repairing the DSBs, and integrating a dsODN at one or more DSBs
Implementation Method 3
integrating a dsODN at one or more DSBs
Data Source
Figure 1
AI summary
Disclosed herein are methods for identifying the effects, including off target effect, of genomic modification of primary cells. These methods are performed with high accuracy suitable for validating therapeutic T-cell engineering approaches.