Miniature Cas14-Derived Editing for Eukaryotic Gene Activation
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Solution Overview
Problem
Existing CRISPR-Cas systems, particularly those involving larger Cas effectors like Cas9 and Cas12a, face challenges in genome engineering applications due to their large size, which limits their use in mammalian cells and systems like adeno-associated virus (AAV) packaging, and there is a need for compact and efficient Cas effectors capable of functioning effectively in eukaryotic cells.
Innovation Solution
Development of engineered miniature Cas effectors, named CasMINI, derived from type V-F Cas14, with optimized amino acid sequences and guide RNA modifications, enabling efficient gene activation and base editing in mammalian cells without off-target effects, and compatible with AAV delivery.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If large Cas effectors (Cas9, Cas12a) are used for genome engineering, then editing efficiency is improved, but packaging capacity in AAV and delivery to mammalian cells is limited
Solution Approach 1:
The patent segments the Cas effector into a miniature version (CasMINI) by deriving it from compact type V-F Cas14 and optimizing its structure. This segmentation allows the effector to fit within AAV packaging capacity while retaining genome editing functionality, resolving the contradiction between size and editing efficiency.
Solution Approach 2:
The patent changes the physical parameter of Cas effector size by engineering CasMINI with optimized amino acid sequences that reduce its volume compared to traditional Cas9/Cas12a. This parameter change enables AAV packaging while maintaining catalytic activity for genome editing in mammalian cells.
2Volume of moving object
If compact Cas effectors (Cas14, CasĪ¦) are used to reduce size, then AAV packaging capacity is improved, but activity in eukaryotic cells is insufficient
Solution Approach 1:
The patent applies parameter changes by optimizing the amino acid sequence of Cas14-derived CasMINI to enhance its catalytic activity in eukaryotic cells. Through iterative engineering, the effector's productivity is improved while maintaining its compact size, enabling both AAV packaging and robust genome editing activity in mammalian cells.
Solution Approach 2:
The patent creates an engineered copy of Cas14 (CasMINI) with optimized properties. This copied and improved version retains the compact size advantage of Cas14 while incorporating mutations that enhance its activity in eukaryotic systems, resolving the activity deficiency of natural compact Cas effectors.
3Volume of moving object
If Cas14 is used for its compact size, then packaging is improved, but gene activation efficiency in mammalian cells is extremely low
Solution Approach 1:
The patent changes the biochemical parameters of Cas14 through protein engineering to dramatically improve gene activation efficiency. The engineered CasMINI exhibits thousands-fold improvement in activation levels compared to wild-type Cas14, while maintaining its compact size for AAV packaging capability.
Data Source
AI summary
Provided herein are Cas proteins and guide RNA molecules engineered to exhibit increased activity in eukaryotic cells. The provided Cas proteins and RNA molecules are particularly useful for applications where modulation of eukaryotic nucleic acids with relatively small molecules is advantageous. Also provided are nucleic acids and vectors encoding the disclosed Cas proteins and guide RNA molecules, pharmaceutical compositions including the Cas proteins and guide RNA molecules, and methods for using the disclosed materials.