Type I-D CAST Genome Insertion for Large DNA Targeting

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Solution Overview

Problem

Current genome-editing technologies lack affordability, ease of setup, scalability, and the ability to target multiple positions within the eukaryotic genome effectively.

Innovation Solution

Development of an engineered system comprising Type I-D Cas proteins, CRISPR-associated Tn7 transposases, and guide molecules for sequence-specific binding and insertion of large polynucleotides into target DNA without requiring strand breaks.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If conventional genome-editing technologies (designer zinc fingers, TALEs, homing meganucleases) are used, then targeted genome perturbations can be produced, but the technologies are expensive, difficult to set up, and not scalable

Engineering Contradiction:

Improvetargeted genome perturbation capabilityVSAvoidease of setup and scalability

Core Design Contradiction:

ReliabilityVSEase of manufacture

Solution Approach 1:

The system divides the genome-editing function into separate modular components: a portable Cas effector unit (Cas proteins) and a guide molecule unit (crRNA/tracrRNA). This segmentation allows independent optimization and simplifies setup, as only the small guide molecules need to be designed for each target site while the Cas proteins remain constant.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The Cas effector proteins serve multiple functions: they provide structural stability, guide target recognition through crRNA binding, and execute the genome perturbation. This multi-functionality reduces the need for separate components and simplifies the overall system setup.

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Reliability

If conventional genome-editing technologies are used, then targeted genome perturbations can be produced, but the technologies are complex and not amenable to targeting multiple positions

Engineering Contradiction:

Improvetargeted genome perturbation capabilityVSAvoidsystem complexity and multi-position targeting capability

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The system uses dynamic RNA-protein interactions where crRNA molecules can be easily exchanged or redesigned to target different positions in the genome. The Cas effector proteins maintain stable structural conformation while adapting their target specificity through RNA binding, enabling flexible multi-position targeting without redesigning the entire protein system.

Inventive Principle:

Principle #15Dynamics

3Measurement precision

If Type I-D CRISPR-Cas systems are used for genome engineering, then precise targeting is achieved, but the systems require complex multi-subunit effector complexes

Engineering Contradiction:

Improvesequence-specific binding precisionVSAvoidmulti-subunit effector complex complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent extracts and simplifies the essential function of Type I-D CRISPR-Cas systems by using minimal Cas effector proteins (Cas5, Cas6, Cas7, Cas10d) that can function with guide molecules. This extraction removes unnecessary complexity while maintaining the core precision of sequence-specific binding through crRNA-guided target recognition.

Inventive Principle:

Principle #2Taking out (Extraction)

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

Enables precise and efficient insertion of donor sequences into target polynucleotides, facilitating genetic perturbations and corrections, and providing a scalable platform for genome engineering and biotechnology applications.

Implementation Method 1

a guide molecule capable of forming a complex with the one or more Type I-D Cas proteins and directing sequence-specific binding of the complex to the target polynucleotide

Methodology Applied

Scientific EffectBase pairing complementarity:

Implementation Method 2

one or more CRISPR-associated Tn7 transposases or functional fragments thereof linked to or otherwise capable of associated with the one or more Type I-D Cas proteins

Methodology Applied

Scientific EffectTransposition:

Data Source

PatentUS20260015631A1Type i-d crispr-associated transposase and tyrosine recombinase transposon systems

Publication Date: 2026.01.15 THE BROAD INST INC

AI summary

The invention provides for systems and methods for inserting large polynucleotides into precise locations in a target polynucleotide. In one aspect, the systems comprise an engineered Type I-D/Tn7 CRISPR-associated transposase system (CAST) comprising a Tn7-like transposase linked to or otherwise capable of associating with a Type I-D CRISPR-Cas complex (Tn7-CAST I-D). In another aspect, the systems comprise a Tn7-like transposase comprising a modular target site selection protein, called TnsF, that may be engineered to reprogram the Tn7-like transposase to facilitate insertion at different sites in a target polynucleotide. In another aspect, the systems comprise a transposon system comprising a tyrosine recombinase which provides for scar-less insertion of large donor sequences into target polynucleotides. Also provided are methods for modifying target polynucleotides using the systems; polynucleotides encoding the systems; delivery systems for delivering the components of the systems; and cells and biological products modified by or modified to include the systems.