gRNA Library Generation via Insertional Enzyme Complexes
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Solution Overview
Problem
Current methods for generating genome-wide CRISPR-Cas knockout screens are resource-intensive and costly, limiting their application to only protein-coding regions of the reference genome, while tools for systematically screening non-coding genomes for functionality are lacking, particularly in genome-wide association studies where most disease-linked single nucleotide polymorphisms are found.
Innovation Solution
A method using insertional enzyme complexes with tagmentation adapters to generate libraries of polynucleotide molecules encoding guide RNAs (gRNAs) that target both coding and non-coding regions, enabling high-throughput and cost-effective patient- and cell-type specific gRNA libraries without prior knowledge of sequence or chromatin profiles.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If universal libraries generated by a handful of labs are used for genome-wide CRISPR screens, then resource intensity and cost are reduced, but the ability to target non-coding genome regions and achieve patient/cell-type specificity is lost
Solution Approach 1:
The method enables laboratories to generate their own customized gRNA libraries in-house using insertional enzyme complexes and tagmentation adapters, eliminating the need to rely on universal libraries from external sources. This self-service approach allows institutions to create patient- and cell-type specific libraries tailored to their research needs while maintaining cost-effectiveness
Solution Approach 2:
The invention changes the fundamental parameters of library generation by using insertional enzyme complexes with tagmentation adapters instead of traditional oligonucleotide synthesis methods. This enables the generation of libraries targeting non-coding regions and allows for patient/cell-type specificity through chromatin accessibility-based enrichment, while maintaining high-throughput and cost-effective characteristics
2Quantity of substance
If traditional oligonucleotide design and synthesis methods are used, then comprehensive genome coverage is achieved, but resource intensity and cost increase significantly
Solution Approach 1:
The invention replaces the mechanical/chemical synthesis process of traditional oligonucleotide design with a biological-based insertional enzyme complex system. The tagmentation adapters and insertional enzymes naturally generate and process gRNA sequences, eliminating the need for resource-intensive synthetic chemistry while achieving comprehensive genome coverage including non-coding regions
Solution Approach 2:
The insertional enzyme complexes with tagmentation adapters serve multiple functions: they fragment DNA, add adapters, and enable generation of gRNAs targeting both coding and non-coding regions. This multi-functional system replaces multiple separate steps in traditional methods, reducing overall resource intensity while maintaining comprehensive genome coverage
3Adaptability or versatility
If tools for screening non-coding genome are developed, then functionality of non-coding regions can be systematically studied, but method complexity and development resources increase
Solution Approach 1:
The method performs preliminary enrichment of open chromatin regions before library generation, using the natural accessibility of chromatin to preferentially capture functional non-coding sequences. This preliminary action simplifies subsequent steps by pre-selecting relevant targets, reducing overall method complexity while enabling systematic study of non-coding genome functionality
Solution Approach 2:
The invention merges chromatin accessibility analysis with gRNA library generation into a single integrated workflow. The insertional enzyme complexes operate directly on chromatin or genomic DNA, combining what would traditionally be separate experiments (chromatin profiling and library construction) into one streamlined process, thereby reducing method 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 allows for the generation of comprehensive gRNA libraries that target all functional regions of the genome, including non-coding areas, capturing unique genetic variants and enriching for open chromatin regions, thereby facilitating more extensive genetic analysis at a lower cost.
Implementation Method 1
incubation of the target polynucleotide(s) with insertional enzyme complexes, wherein each of said insertional enzyme complexes comprises (i) an insertional enzyme and (ii) one or more tagmentation adapters to generate a plurality of tagged cleavage fragments
Data Source
AI summary
The invention relates to a method of generating a library of polynucleotide molecules encoding guide RNAs (gRNAs) from target polynucleotide(s). The invention also relates to a library of polynucleotide molecules encoding gRNAs obtainable by the aforementioned method, and a gRNA library generation kit thereof.