Guide RNA Sequence Variant Detection via ds-cDNA Sequencing
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Solution Overview
Problem
Current methods for assessing guide RNA (gRNA) samples, such as mass spectrometry, fail to identify contaminants like nucleotide substitutions, insertions, or deletions, which can cause off-target effects and hinder clinical applications.
Innovation Solution
The method involves generating a library of double-stranded complementary DNA (ds-cDNA) molecules from gRNAs, sequencing them, and aligning the sequences to a reference to determine levels of truncations, insertions, deletions, and substitutions, allowing for the identification of sequence variants and potential off-target sites.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If mass spectrometry methods are used to assess gRNA samples, then the assessment process is simplified, but the ability to identify contaminants such as nucleotide substitutions, insertions, or deletions is lost
Solution Approach 1:
The patent replaces mass spectrometry (a physical/chemical analysis method) with next-generation sequencing technology. This substitution enables precise identification of nucleotide-level variations including substitutions, insertions, and deletions in gRNA samples, thereby resolving the contradiction between operational simplicity and measurement precision by using a more advanced analytical platform that provides both ease of use and high detection accuracy
Solution Approach 2:
The patent changes the detection parameter from mass-to-charge ratio (used in mass spectrometry) to nucleotide sequence information. By sequencing the gRNA molecules and comparing them to the reference sequence, the method can precisely identify contaminants at the nucleotide level, achieving both operational feasibility and high measurement precision for contaminant detection
2Measurement precision
If next-generation sequencing is used to assess gRNA samples, then the detection of sequence variants is improved, but the complexity of the assessment system increases
Solution Approach 1:
The patent segments the complex sequencing assessment into distinct functional modules: (1) gRNA sample preparation and conversion to cDNA, (2) next-generation sequencing, (3) sequence alignment to reference, and (4) contaminant identification and quantification. This segmentation allows each module to be optimized independently and simplifies the overall system by breaking down the complex assessment process into manageable, standardized steps
Solution Approach 2:
The patent introduces double-stranded cDNA as an intermediary between the gRNA sample and the sequencing analysis. The gRNAs are first converted to ds-cDNA molecules, which serve as a stable, sequenceable intermediate that preserves the original gRNA sequence information while being compatible with standard next-generation sequencing platforms, thereby reducing system complexity
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 enables the validation of gRNA sequence integrity and reduces the risk of off-target effects by accurately assessing the presence of contaminants, thereby improving the reliability and safety of gRNA-based therapies.
Implementation Method 1
The step of generating the library of ds-cDNA molecules includes contacting the gRNA composition with a reverse transcriptase, thereby generating a plurality of first strands of the ds-cDNA
Data Source
AI summary
The present disclosure relates to methods of assessing a sample of guide RNAs (gRNAs).