Targeted Nucleic Acid Library Preparation via Programmable Transposome
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Solution Overview
Problem
Conventional library preparation methods for sequencing nucleic acids are time-consuming and produce randomly fragmented libraries, making it difficult to control the output and requiring additional steps like amplicon sequencing or target capture, which are costly and resource-intensive.
Innovation Solution
The use of protein complexes comprising a transposome and a programmable DNA binding unit, such as a nuclease-deficient CRISPR-associated protein (dCAS protein) with a guide RNA, to specifically bind to target double-stranded DNA (dsDNA) and facilitate targeted fragmentation and sequencing library generation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional library preparation methods are used, then libraries can be prepared using standard protocols, but the process takes hours and produces randomly fragmented libraries making it difficult to control the output
Solution Approach 1:
The patent uses CRISPR-Cas proteins as intermediary molecules to guide the fragmentation process. The guide RNA acts as a mediator that directs the Cas protein to specific genomic locations, enabling precise control over which regions are fragmented and sequenced, thereby resolving the contradiction between control precision and time efficiency
Solution Approach 2:
The method performs preliminary targeting by using guide RNAs to pre-identify and bind to specific genomic regions before fragmentation occurs. This preliminary action ensures that only the desired loci are fragmented and included in the sequencing library, eliminating the need for subsequent enrichment steps and reducing overall preparation time
2Manufacturing precision
If amplicon sequencing is used for targeted sequencing, then specific areas can be amplified, but additional amplification steps add costs, time, and resources
Solution Approach 1:
The patent merges the fragmentation and targeting functions into a single integrated step. The CRISPR-Cas system simultaneously performs site-specific DNA cleavage and enrichment of target regions, combining what would traditionally require separate amplification and capture steps into one unified process, thereby reducing device complexity while maintaining targeted sequencing accuracy
Solution Approach 2:
The method extracts only the necessary functional elements (guide RNA-Cas protein complex) needed for targeted fragmentation, eliminating the need for additional amplification reagents and equipment. This extraction of essential functions reduces the number of processing steps while preserving the ability to accurately sequence specific regions
3Manufacturing precision
If target capture method is used for targeted sequencing, then specific nucleic acid targets can be isolated, but the hybridization process is time-consuming and probes are expensive to synthetize
Solution Approach 1:
The patent replaces the mechanical hybridization process with a programmable enzymatic system. Instead of relying on thermal hybridization kinetics and physical isolation methods, the CRISPR-Cas system uses programmable RNA-DNA base pairing combined with enzymatic cleavage to achieve rapid and specific target isolation, eliminating the time-consuming hybridization step while maintaining high specificity
Solution Approach 2:
The method changes the fundamental parameter of target recognition from thermal hybridization to programmable enzymatic binding. By using guide RNA sequences that are complementary to target regions, the system achieves specific binding without requiring prolonged hybridization times or expensive probe synthesis, thereby reducing both time and cost while maintaining isolation specificity
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 rapid targeted sequencing, reducing the time required for library preparation to minutes and allowing for precise control over the sequencing process, thereby improving efficiency, cost-effectiveness, and precision in diagnostic and theranostic applications.
Implementation Method 1
a programmable DNA binding unit capable of specifically binding to a binding site on a target double-stranded DNA (dsDNA)
Implementation Method 2
The transposome comprises a transposase, a first adaptor and a second adaptor
Data Source
AI summary
Disclosed herein include methods, compositions, and kits suitable for use in generating libraries for nucleic acid sequencing. There are provided, in some embodiments, a plurality of protein complexes. Each protein complex can comprise a transposome and a programmable DNA binding unit capable of specifically binding to a user-selected binding site on a target double-stranded DNA (dsDNA). The binding site for each of the plurality of protein complexes can be different from each other. The transposome can comprise a transposase, a first adaptor, and a second adaptor. The first adaptor, the second adaptor, or both, can be a sequencing adaptor.