CRISPR-Cas Functional Genomics Screening for Precise Gene Knockouts

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Solution Overview

Problem

Current functional genomics screens face challenges with RNAi technologies due to off-target effects, limited spatial control, and the need for gene knockdown rather than knockout, necessitating affordable, scalable, and controlled genome engineering methods.

Innovation Solution

The CRISPR-Cas system is employed for gene targeting and knockout, utilizing programmable short RNA molecules to recognize specific DNA targets, enabling parallel targeting of thousands of genomic loci through oligo library synthesis and vector delivery systems like AAV or lentiviral vectors.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If RNAi technology is used for functional genomics screens, then gene knockdown can be achieved, but off-target effects occur and spatial control is limited

Engineering Contradiction:

Improvegene knockdown efficacyVSAvoidoff-target effects

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The patent replaces RNAi-based gene knockdown (mechanical/biochemical system) with CRISPR-Cas9 genome editing system. The CRISPR-Cas9 system uses guide RNA to direct Cas9 nuclease to specific DNA sequences, creating precise gene knockouts without the off-target effects characteristic of RNAi. This substitution fundamentally changes the mechanism from RNA-mediated interference to DNA-targeted cleavage and repair.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Reliability

If RNAi technology is used for functional genomics screens, then gene expression can be reduced, but spatial control is limited

Engineering Contradiction:

Improvegene expression controlVSAvoidspatial control

Core Design Contradiction:

ReliabilityVSEase of operation

Solution Approach 1:

The patent implements local quality by enabling spatially controlled gene editing through the CRISPR-Cas9 system. By delivering Cas9 and guide RNA to specific locations or using inducible systems, the invention achieves precise spatial control over where and when gene editing occurs, allowing different regions or cell types to be edited independently based on local requirements.

Inventive Principle:

Principle #3Local quality

3Manufacturing precision

If CRISPR-Cas system is used for genome engineering, then gene knockout precision is improved, but method complexity increases

Engineering Contradiction:

Improvegene knockout precisionVSAvoidmethod complexity

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The patent applies universality by creating a standardized CRISPR-Cas9 platform that can target any gene sequence through simple guide RNA design. The system uses universal components (Cas9 nuclease, guide RNA structure, PAM sequence requirements) that can be applied across different genes and experimental contexts, reducing the need for gene-specific optimization and simplifying the overall methodology despite the precision achieved.

Inventive Principle:

Principle #6Universality (Multi-functionality)

4Ease of manufacture

If functional genomics screens are performed with traditional methods, then established protocols can be used, but productivity is limited

Engineering Contradiction:

Improveprotocol establishmentVSAvoidscreening throughput

Core Design Contradiction:

Ease of manufactureVSProductivity

Solution Approach 1:

The patent implements continuous useful action by enabling high-throughput functional genomics screening through CRISPR-Cas9. The system allows parallel processing of multiple genes simultaneously through library-based approaches, where pools of guide RNAs targeting different genes can be delivered and screened in a single experiment, maintaining continuous productive action without the sequential limitations of traditional methods.

Inventive Principle:

Principle #20Continuity of useful action

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, allowing for robust gene knockouts with minimal off-target activity, facilitating the cataloging and mapping of genetic factors associated with diverse 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

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

the CRISPR enzyme cleaves the genomic loci of the DNA molecule encoding the gene product

Methodology Applied

Scientific EffectEnzymatic cleavage: Enzyme

Data Source

PatentUS12441995B2Functional genomics using CRISPR-Cas systems, compositions, methods, screens and applications thereof

Publication Date: 2025.10.14 THE BROAD INST INC

AI summary

The present invention generally relates to libraries, compositions, methods, applications, kits 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. Provided are vectors and vector systems, some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for utilizing the CRISPR-Cas system.