Cas9-gRNA Targeted Nucleic Acid Enrichment
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for nucleic acid complexity reduction in genetic research lack flexibility and accuracy, particularly in enriching target nucleic acid fragments for subsequent analysis, and often require amplification or additional protection steps.
Innovation Solution
A method involving the use of RNA or DNA guided endonucleases, such as gRNA-CAS complexes, to cleave nucleic acid molecules, followed by exonuclease treatment to digest non-target fragments, allowing for the enrichment and purification of target nucleic acid fragments without the need for additional protection or amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If type II restriction enzymes are used to fragment nucleic acids followed by ligation of protective adapters and degradation of non-captured nucleic acid using exonucleases, then complexity reduction is achieved, but the method requires additional protection steps and amplification
Solution Approach 1:
The invention extracts and utilizes the protective function of the Cas9-gRNA complex itself, eliminating the need for separate protective adapters. The Cas9-gRNA complex remains bound to the target fragment throughout the process, providing continuous protection while enabling direct enrichment without additional protection steps.
Solution Approach 2:
The Cas9-gRNA complex serves multiple functions simultaneously: it acts as both a protective element (preventing exonuclease digestion) and a targeting element (guiding the enrichment process). This multi-functionality eliminates the need for separate protective adapters and reduces the number of required steps.
2Measurement precision
If protective adapters are ligated to protect target nucleic acid fragments, then enrichment accuracy is improved, but the method requires amplification and additional processing steps
Solution Approach 1:
The Cas9-gRNA complex provides self-protection by remaining bound to the target fragment, eliminating the need for external protective adapters. This self-service mechanism maintains enrichment accuracy while eliminating unnecessary processing steps including amplification.
Solution Approach 2:
The Cas9-gRNA complex acts as an intermediary that directly protects the target fragment without requiring protective adapters as intermediaries. This direct protection mechanism simplifies the workflow by eliminating the ligation and amplification steps while maintaining accuracy.
3Quantity of substance
If amplification is performed to enrich target nucleic acid fragments, then the amount of target material is increased, but the method introduces bias and reduces accuracy
Solution Approach 1:
The invention extracts the protective function from separate protective adapters and integrates it into the Cas9-gRNA complex itself. This eliminates the need for amplification while maintaining sufficient target material for downstream applications, thereby avoiding amplification-induced bias and maintaining accuracy.
4Productivity
If multiple processing steps including ligation and amplification are performed, then enrichment efficiency is improved, but the overall process time and complexity increase
Solution Approach 1:
The invention merges the protective function and targeting function into the single Cas9-gRNA complex, eliminating the need for separate protective adapters and their ligation steps. This consolidation maintains enrichment efficiency while significantly reducing the overall process time by eliminating unnecessary steps.
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 simplifies library preparation, reduces sample complexity, and enhances the accuracy of nucleic acid enrichment, enabling efficient downstream processing and analysis while eliminating the need for amplification or protective adapters.
Implementation Method 1
b) cleaving the nucleic acid molecule with at least a first and a second RNA or DNA guided endonuclease complex, thereby generating the target nucleic acid fragment comprising the sequence of interest and at least one non-target nucleic acid fragment
Implementation Method 2
c) contacting the cleaved nucleic acid molecules obtained in step b) with an exonuclease and allowing the exonuclease to digest the at least one non-target nucleic acid fragment
Data Source
AI summary
The current invention pertains to a method for the enrichment of a target nucleic acid fragment from a nucleic acid sample, comprising the steps of cleaving the nucleic acid sample with a first and a second RNA guided or DNA guided endonuclease complex, preferably a first and a second gRNA-CAS complex, thereby generating the target nucleic acid fragment and at least one non-target nucleic acid fragment. The generated fragments are subsequently contacted with an exonuclease, wherein the exonuclease digests only the non-target nucleic acid fragments. The invention further pertains to the use of the enriched target nucleic acid fragments for preparing an adapter ligated target nucleic acid fragment and for sequencing the target nucleic acid fragment.