Compact Type II Cas Proteins for AAV Packaging

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Solution Overview

Problem

The limited packaging capacity of adeno-associated viral vectors (AAVs) makes it challenging to package large Cas proteins like SpCas9 together with guide RNAs in a single vector, limiting the efficiency of CRISPR-Cas genome editing.

Innovation Solution

The discovery and development of smaller Type II Cas proteins, such as BNK, AIK, HPLH, and ANAB Type II Cas proteins, which are approximately 1000 amino acids in length, allowing for more efficient packaging within AAV vectors, along with the use of guide RNAs to direct genome editing.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If large Cas proteins like SpCas9 are used for CRISPR-Cas genome editing, then editing functionality is achieved, but packaging capacity of AAV vectors is exceeded

Engineering Contradiction:

Improvegenome editing functionalityVSAvoidCas protein size

Core Design Contradiction:

Adaptability or versatilityVSQuantity of substance

Solution Approach 1:

The patent changes the size parameter of the Cas protein by using alternative Type II Cas proteins (such as CjCas9, Nme2Cas9, SaCas9, or StCas9) that are smaller than SpCas9. These alternative Cas proteins have different amino acid lengths and structural properties that allow them to be packaged within AAV vectors while maintaining CRISPR-Cas editing functionality.

Inventive Principle:

Principle #35Parameter changes

2Ease of operation

If AAV vectors are used to deliver Cas proteins and guide RNAs, then delivery capability is achieved, but packaging capacity is limited

Engineering Contradiction:

Improvevector delivery capabilityVSAvoidpackaging capacity

Core Design Contradiction:

Ease of operationVSQuantity of substance

Solution Approach 1:

The patent changes the parameter of Cas protein size to match the packaging capacity constraints of AAV vectors. By selecting smaller Type II Cas proteins, the total payload size (Cas protein + guide RNA) remains within the acceptable packaging capacity range of AAV vectors, enabling successful delivery.

Inventive Principle:

Principle #35Parameter changes

3Quantity of substance

If smaller Type II Cas proteins are used, then packaging capacity is improved, but protein functionality may be compromised

Engineering Contradiction:

ImproveCas protein sizeVSAvoidprotein editing functionality

Core Design Contradiction:

Quantity of substanceVSReliability

Solution Approach 1:

The patent employs alternative Type II Cas proteins that are structurally and functionally optimized for their size. These smaller Cas proteins (CjCas9, Nme2Cas9, SaCas9, StCas9) have been characterized to ensure they maintain essential nuclease activity, PAM recognition, and guide RNA binding capabilities despite their reduced size compared to SpCas9.

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

Data Source

PatentUS20250197854A1Type ii cas proteins and applications thereof

Publication Date: 2025.06.19 ALIA THERAPEUTICS SRL

AI summary

Type II Cas proteins, for example Type II Cas proteins referred to as AIK Type II Cas proteins, BNK Type II Cas proteins, HPLH Type II Cas proteins, and ANAB Type II Cas proteins; gRNAs for Type II Cas proteins; systems comprising Type II Cas proteins and gRNAs; nucleic acids encoding the Type II Cas proteins, gRNAs and systems; particles comprising the foregoing; pharmaceutical compositions of the foregoing; and uses of the foregoing, for example to alter the genomic DNA of a cell.