Guide RNA Sequencing via Modified Template Switching Oligonucleotides

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Current methods for assessing the purity and sequence of guide RNAs, such as electrospray ionization and high-performance liquid chromatography, have low sensitivity and cannot resolve the sequence composition of single-guide RNA molecules, leading to issues with off-target genome editing and varied editing efficiency in CRISPR/Cas systems.

Innovation Solution

The use of modified template switching oligonucleotides (TSOs) with 3' end modifications, like 3-dideoxycytosine, to prevent DNA polymerase extension, allowing for the synthesis of full-length cDNA copies of guide RNAs, which are then amplified and sequenced to determine their sequence relative to a reference guide RNA sequence.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If electrospray ionization and high-performance liquid chromatography are used to assess guide RNA purity, then the method is simple and widely applicable, but the sensitivity in detecting impurities is relatively low (1-2%) and cannot resolve the sequence composition of single-guide RNA molecules

Engineering Contradiction:

Improvepurity detection sensitivityVSAvoidsequencing method complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent uses a template switching oligonucleotide (TSO) as an intermediary molecule to enable sequencing of guide RNAs. The TSO contains a 3' terminal modification that prevents DNA polymerase extension, allowing it to serve as a switching template during cDNA synthesis. This intermediary approach enables high-resolution sequence composition analysis while maintaining practical laboratory implementation.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent modifies the chemical structure of the TSO by adding specific modifications at the 3' terminal end (such as 3-dideoxycytosine) to change its properties. This parameter change prevents DNA polymerase extension and enables the TSO to function as a template switch, thereby achieving high sensitivity in impurity detection and sequence composition resolution.

Inventive Principle:

Principle #35Parameter changes

2Measurement precision

If next-generation sequencing methods are used to sequence guide RNAs, then sequence composition can be resolved, but the methods are prone to mis-priming and generation of artifacts that interfere with reliable outcomes

Engineering Contradiction:

Improvesequence composition resolutionVSAvoidsequencing accuracy

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

The patent applies preliminary anti-action by incorporating a 3' terminal modification into the TSO that specifically prevents DNA polymerase extension. This pre-established protective mechanism stops the formation of artifacts and mis-priming events before they can occur during the sequencing process, thereby ensuring reliable sequencing outcomes.

Inventive Principle:

Principle #9Preliminary anti-action

Solution Approach 2:

The patent converts the potentially harmful effect of DNA polymerase activity into a beneficial control mechanism. By using the 3' terminal modification to selectively prevent extension, the method transforms what could be a source of errors into a feature that ensures high sequencing accuracy and reliability.

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

3Productivity

If CRISPR/Cas systems are used for gene editing, then genome editing can be achieved, but unwanted off-target genome editing and varied editing efficiency occur

Engineering Contradiction:

Improvegene editing efficiencyVSAvoidoff-target editing

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent implements feedback by providing a sequencing method that allows monitoring and verification of guide RNA sequence composition. This feedback mechanism enables detection of off-target effects and editing efficiency variations, allowing for quality control and optimization of CRISPR/Cas systems to reduce harmful off-target editing.

Inventive Principle:

Principle #23Feedback

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 method enhances the accuracy of guide RNA sequencing, reducing artifacts and mis-priming, and achieves error correction to less than 1%, ensuring high purity and identity of guide RNA compositions, thereby improving the reliability of CRISPR/Cas systems.

Implementation Method 1

DNA primer that hybridizes to the 3' end of the guide RNA

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

synthesis of cDNA from the guide RNA template using a primer specific for the 3' end of the guide RNA

Methodology Applied

Scientific EffectReverse transcription:

Implementation Method 3

a template switching oligonucleotide (TSO) comprising a modification at the 3' terminal end... the modification is a 3-dideoxycytosine (3ddC)... the 3' end modification on the TSO prevents second strand extension during PCR amplification

Methodology Applied

Scientific EffectPolymerase blocking:

Implementation Method 4

amplifying the cDNA... the cDNA is amplified by PCR

Methodology Applied

Scientific EffectPCR amplification:

Implementation Method 5

sequencing the amplified cDNA... the sequencing comprises NGS

Methodology Applied

Scientific EffectNext-generation sequencing:

Data Source

PatentUS20240368586A1Guide RNA sequencing confirmation

Publication Date: 2024.11.07 SYNTHEGO CORP

AI summary

Provided herein are methods for assessing a preparation of guide RNA molecules for quality, including for purity and identity.