CRISPR-Cas9 sgRNA Library Design for Genome-Wide Knockout
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Solution Overview
Problem
Current functional genomics methods, such as those using RNA interference (RNAi), face limitations including off-target effects, spatial control issues, and the need for continuous expression of shRNAs, necessitating the development of more efficient and robust genome engineering technologies for gene knockout applications.
Innovation Solution
The CRISPR-Cas system is employed to target specific DNA sequences using short RNA molecules, enabling parallel targeting of thousands of genomic loci without the need for customized proteins, and is integrated into vector systems for genome-wide knockout capabilities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If RNA interference (RNAi) is used for functional genomics, then gene expression can be suppressed, but off-target effects occur and spatial control is limited
Solution Approach 1:
The patent replaces the RNAi mechanism with the CRISPR-Cas9 system, substituting one molecular mechanism for another more precise one. CRISPR-Cas9 uses guide RNA to direct Cas9 nuclease to specific DNA sequences for cleavage, providing more precise gene knockout capability compared to RNAi's gene suppression approach, thereby reducing off-target effects while maintaining reliable gene function disruption
Solution Approach 2:
The patent changes the molecular parameter from RNA-based gene suppression (RNAi) to DNA-based gene knockout (CRISPR-Cas9). This fundamental parameter change enables permanent genetic modification rather than transient suppression, improving reliability and reducing off-target effects through precise DNA targeting and cleavage followed by error-prone repair
2Reliability
If short hairpin RNAs (shRNAs) are used for gene silencing, then gene expression is suppressed, but continuous expression is required maintaining complexity
Solution Approach 1:
The patent implements preliminary action by performing permanent DNA modification through CRISPR-Cas9-mediated cleavage and repair. Unlike shRNA that requires continuous transcription and translation to maintain silencing, CRISPR-Cas9 creates permanent genetic changes that persist through cell divisions without requiring continuous expression of the machinery, thereby simplifying the system while maintaining reliable gene knockout
Solution Approach 2:
The patent uses the cell's own DNA repair machinery as a copy mechanism. After CRISPR-Cas9 creates double-strand breaks, the cell's error-prone non-homologous end joining (NHEJ) repair pathway copies the disruption permanently into the genome, eliminating the need for continuous shRNA expression and reducing system complexity
3Measurement precision
If customized proteins are used for genome targeting, then specific genomic loci can be targeted, but device complexity increases
Solution Approach 1:
The patent applies universality by using a single Cas9 nuclease protein that can target any genomic locus when guided by appropriately designed guide RNA sequences. This eliminates the need for customized proteins for each target site, as the same Cas9 enzyme performs all targeting functions through RNA guidance, thereby maintaining high precision while reducing device complexity
Solution Approach 2:
The patent introduces guide RNA as an intermediary between the Cas9 nuclease and the target DNA sequence. This intermediary carries the targeting information and directs Cas9 to the correct genomic locus without requiring customization of the Cas9 protein itself, achieving precise targeting while simplifying the overall system architecture
4Loss of information
If genome-wide screening is performed, then comprehensive genetic factor mapping is achieved, but time consumption increases
Solution Approach 1:
The patent creates an inert environment for high-throughput screening by using pooled CRISPR-Cas9 libraries where multiple guide RNAs targeting different genes are delivered simultaneously to cell populations. This enables parallel screening of thousands of genetic factors in a single experiment rather than sequential analysis, dramatically reducing time while maintaining comprehensive coverage through the inert, controlled library delivery system
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 functional genomics methodologies, provides precise gene knockout with minimal off-target activity, and offers spatial and temporal control, accelerating the mapping of genetic factors associated with biological functions and diseases.
Implementation Method 1
the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in the genomic loci of the DNA molecule encoding the gene product
Implementation Method 2
the CRISPR enzyme cleaves the genomic loci of the DNA molecule encoding the gene product
Data Source
AI summary
The present invention generally relates to libraries, kits, methods, applications and screens used in functional genomics that focus on gene function in a cell and that may use vector systems and other aspects related to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas systems and components thereof. The present invention also relates to rules for making potent single guide RNAs (sgRNAs) for use in CRISPR-Cas systems. Provided are genomic libraries and genome wide libraries, kits, methods of knocking out in parallel every gene in the genome, methods of selecting individual cell knock outs that survive under a selective pressure, methods of identifying the genetic basis of one or more medical symptoms exhibited by a patient, and methods for designing a genome-scale sgRNA library.