Hypercompact CRISPR/Cas12f Target System for AAV-Compatible HDR

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Solution Overview

Problem

Current gene editing technologies face limitations due to low homology-directed repair (HDR) efficiency and the inability to be packaged efficiently within adeno-associated virus (AAV) vectors, which are essential for intracellular delivery.

Innovation Solution

A hypercompact CRISPR/Cas12f nucleic acid editing system comprising Cas12f1, TnpB, or variant proteins, engineered guide RNAs, and donor nucleic acids, which can be packaged into AAV vectors, enhancing HDR efficiency and incorporating inhibitors of non-homologous end joining.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If conventional CRISPR/Cas9 systems are used for gene editing, then gene editing capability is achieved, but the system size exceeds the packaging capacity of AAV vectors (4.7 kb)

Engineering Contradiction:

ImproveAAV vector packaging compatibilityVSAvoidsystem size

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The patent extracts and replaces the large Cas9 protein with a significantly smaller Cas12f1 protein (approximately 1/3 the size), enabling the gene editing system to fit within AAV vector packaging capacity while retaining core gene editing functionality

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent uses engineered guide RNA molecules that are shorter in length than conventional guides, creating a compact version of the targeting mechanism that works efficiently with the miniaturized Cas12f1 system within AAV constraints

Inventive Principle:

Principle #26Copying

2Manufacturing precision

If non-homologous end joining (NHEJ) is the default repair mechanism, then DNA breaks are repaired quickly, but precise gene editing through homology-directed repair (HDR) has low efficiency

Engineering Contradiction:

ImproveHDR efficiencyVSAvoidrepair time

Core Design Contradiction:

Manufacturing precisionVSLoss of time

Solution Approach 1:

The patent incorporates inhibitors of non-homologous end joining (NHEJ) into the system, which preemptively block the dominant but imprecise repair pathway, thereby allowing homology-directed repair to proceed with higher efficiency and precision

Inventive Principle:

Principle #9Preliminary anti-action

Solution Approach 2:

The patent introduces donor nucleic acid molecules as intermediaries that provide the template for precise gene editing through HDR, enabling accurate sequence insertion or modification at the target site

Inventive Principle:

Principle #24Intermediary (Mediator)

Data Source

PatentUS20250215457A1Target system for homology-directed repair and gene editing method using same

Publication Date: 2025.07.03 GENKORE INC

AI summary

The present invention relates to a novel target nucleic acid editing system including a miniaturized nucleic acid editing protein and an engineered guide RNA, uses of homology-directed repair (HDR) in a target gene thereof, and the like. According to one embodiment, homology-directed repair (HDR) using the target nucleic acid editing system has high HDR efficiency compared to other CRISPR/Cas systems due to a tendency to cut the back (outside) of a target nucleic acid, and also has the effect of enabling packaging of a gene editing system including a donor nucleic acid in a single vector, even when the packaging size is very limited like adeno-associated viruses (AAV), and maximizing the HDR efficiency by adding shRNA that inhibits a non-homologous end joining (NHEJ) process.