CRISPR Saturating Mutagenesis of Noncoding Regions in Native Chromatin
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Solution Overview
Problem
Current methods lack the ability to systematically interrogate noncoding genomic regions for functional elements that affect phenotypes and gene regulation within a native biological context, as existing tools are biased and decoupled from local chromatin interactions.
Innovation Solution
A high-throughput method using pooled CRISPR-Cas9 libraries is employed to screen noncoding genomic loci, enabling unbiased mutagenesis and identification of functional elements by targeting multiple genomic sequences with CRISPR-Cas system guide RNAs, which saturate genomic regions without prior knowledge of the target region.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If existing tools are used to screen noncoding genomic regions, then the screening process is simplified, but the results are biased and decoupled from local chromatin interactions
Solution Approach 1:
The patent introduces an intermediary system (CRISPR-Cas9 with guide RNA library) that mediates between the screening process and the genomic regions. This intermediary enables direct targeting and mutagenesis of noncoding elements within their native chromatin context, eliminating the bias of existing tools while maintaining operational feasibility through high-throughput screening capabilities
2Measurement precision
If comprehensive mutagenesis of noncoding regions is performed, then functional elements are fully identified, but the complexity of the screening method increases
Solution Approach 1:
The patent employs a universal CRISPR-Cas9 system that can target any noncoding genomic region through programmable guide RNAs. This multi-functional approach allows the same core machinery to perform comprehensive mutagenesis across diverse genomic loci, achieving high measurement precision without proportionally increasing device complexity
Solution Approach 2:
The patent utilizes parameter changes in the guide RNA sequence to direct the Cas9 enzyme to different genomic targets. By varying the guide RNA sequence parameters while maintaining the core CRISPR-Cas9 system, comprehensive mutagenesis is achieved with minimal increase in system complexity
3Measurement precision
If saturation mutagenesis is applied to noncoding elements, then all functional variants are discovered, but the time and resources required increase significantly
Solution Approach 1:
The patent applies saturation mutagenesis strategically to noncoding elements rather than uniformly across the entire genome. By focusing mutagenesis on predicted or suspected functional regions identified through preliminary analysis, the method achieves comprehensive validation of functional variants while reducing overall screening time and resource requirements
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 discovery of both gene-proximal and gene-distal functional elements, providing comprehensive insights into gene regulation and phenotypic changes, including cancer drug resistance, by saturating mutagenesis and validating genomic sites associated with phenotypic changes.
Implementation Method 1
CRISPR-Cas9-mediated gene disruption has been widely used in generating loss-of-function mutations
Implementation Method 2
the guide RNAs, which direct sequence-specific binding of a CRISPR-Cas system to target sequences
Data Source
AI summary
The application relates to a deep scanning mutagenesis library to interrogate phenotypic changes in a population of cells comprising a plurality of CRISPR-Cas system guide RNAs targeting genomic sequences within at least one continuous genomic region, wherein the guide RNAs target at least 100 genomic sequences upstream of a PAM sequence for every 1000 base pairs within the continuous genomic region and methods for their use.