Large Targeting Vector CRISPR Rat Genome Editing

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Solution Overview

Problem

The use of rats in modeling human diseases is limited due to the unavailability of germline-transmittable pluripotent rat cells and inefficient targeting technologies for introducing or deleting large genomic DNA sequences in rat cells, hindering precise targeted genetic modifications and therapeutic agent validation.

Innovation Solution

A method involving a large targeting vector (LTVEC) with 5' and 3' homology arms, combined with Cas protein and CRISPR RNA, allows for targeted genetic modifications in rat cells, enabling precise changes to the genome, including biallelic modifications, deletions, and insertions of exogenous sequences, facilitating therapeutic agent validation and disease modeling.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If conventional targeting technologies are used in rat cells, then the process is simple, but the precision and efficiency of targeted genetic modification is poor

Engineering Contradiction:

Improveprecision of targeted genetic modificationVSAvoidcomplexity of targeting technology

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The patent combines CRISPR/Cas9 system with large targeting vectors (LTVECs) to merge the precision of CRISPR-based targeting with the capacity for large-scale genomic modifications. This integration enables simultaneous achievement of precise targeted modification and large sequence insertion/deletion capabilities that were previously mutually exclusive in conventional systems.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The patent introduces LTVECs as intermediary carriers that facilitate targeted genetic modification in rat cells. These vectors serve as mediators between the CRISPR/Cas9 system and the target genome, enabling efficient delivery and integration of large genomic sequences while maintaining precision through homology-directed recombination mechanisms.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Adaptability or versatility

If large genomic DNA sequences are introduced or deleted, then the functional capability is improved, but the efficiency of targeting is reduced

Engineering Contradiction:

Improvefunctional capability for disease modelingVSAvoidefficiency of targeting

Core Design Contradiction:

Adaptability or versatilityVSProductivity

Solution Approach 1:

The patent changes the size parameter of targeting vectors by utilizing large targeting vectors (LTVECs) that can accommodate substantial genomic sequences. This parameter change enables the system to handle large-scale modifications while maintaining high targeting efficiency through optimized homology arm designs and CRISPR-mediated double-strand break formation at precise locations.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent employs preliminary action by pre-designing homology arms and CRISPR target sites before introducing the LTVEC into rat cells. This preliminary planning ensures that when the vector enters the cell, the targeting machinery can immediately initiate precise modification without requiring subsequent selection or screening steps, thereby maintaining high efficiency despite the large size of the genomic sequences involved.

Inventive Principle:

Principle #10Preliminary action

3Adaptability or versatility

If pluripotent rat cells are made available, then the versatility for disease modeling is improved, but the availability and sustainability of such cells is currently limited

Engineering Contradiction:

Improveversatility for disease modelingVSAvoidavailability and sustainability of pluripotent cells

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The patent creates a universal system that can be applied across different rat cell types and disease models. By establishing a standardized protocol combining LTVEC delivery with CRISPR/Cas9 activation, the method achieves multi-functionality for various applications including gene knockouts, knockins, and large-scale genomic rearrangements, thereby improving availability and sustainability of pluripotent cell work for disease modeling.

Inventive Principle:

Principle #6Universality (Multi-functionality)

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 method enables efficient and precise genetic modifications in rat cells, expanding the capabilities for disease modeling and therapeutic agent validation, thereby overcoming the limitations of current technologies.

Implementation Method 1

a third nucleic acid encoding a guide RNA (gRNA) comprising a nucleotide sequence that hybridizes to a target sequence

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

a first expression construct comprising a first promoter operably linked to a second nucleic acid encoding a Cas protein

Methodology Applied

Scientific EffectEnzymatic cleavage: Enzyme

Data Source

PatentUS20240052365A1Methods and compositions for the targeted modification of a genome

Publication Date: 2024.02.15 REGENERON PHARMACEUTICALS INC

AI summary

Compositions and methods are provided for modifying a genomic locus of interest in a eukaryotic cell, a mammalian cell, a human cell or a non-human mammalian cell using a large targeting vector (LTVEC) comprising various endogenous or exogenous nucleic acid sequences as described herein. Further methods combine the use of the LTVEC with a CRISPR/Cas system. Compositions and methods for generating a genetically modified non-human animal comprising one or more targeted genetic modifications in their germline are also provided.