Miniature CasMINI CRISPR Engineering for AAV-Ready Genome Editing
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Solution Overview
Problem
Existing CRISPR-Cas systems face challenges due to their large size, which limits their application in mammalian cells, particularly in gene therapy and cell engineering, and compact and efficient Cas effectors are needed for effective genome editing.
Innovation Solution
Development of engineered miniature Cas effectors, such as CasMINI, derived from type V-F Cas14, with optimized amino acid sequences and guide RNA designs, enabling efficient gene activation and base editing in mammalian cells without off-target effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional CRISPR-Cas systems (Cas9, Cas12a) are used for genome editing, then editing efficiency and specificity are achieved, but the large protein size prohibits applications in mammalian cells and limits delivery capacity
Solution Approach 1:
The patent extracts and utilizes only the essential catalytic domain (RuvC domain) of the Cas14 effector protein, removing unnecessary structural components. This extraction creates a minimal functional unit that retains genome editing capability while dramatically reducing protein size, enabling delivery to mammalian cells through AAV vectors that have strict payload capacity limits.
Solution Approach 2:
The patent applies protein engineering to modify the amino acid sequence of the Cas14 effector, optimizing specific regions (such as the RuvC domain) to enhance catalytic activity and PAM recognition. These parameter changes in the protein sequence enable the miniaturized effector to achieve editing efficiency comparable to or exceeding conventional Cas systems despite the reduced size.
2Weight of stationary object
If compact Cas effectors (Cas14, CasΦ) are used to reduce protein size, then delivery capability is improved, but natural Cas14 shows no activity in mammalian cells and CasΦ exhibits only moderate activity
Solution Approach 1:
The patent applies local quality optimization by focusing protein engineering efforts on specific functional regions of the Cas14 effector, particularly the RuvC domain and PAM recognition motifs. By locally optimizing these critical regions while maintaining the compact overall structure, the engineered effector achieves high activity in mammalian cells without increasing the protein size beyond the AAV delivery limit.
Solution Approach 2:
The patent creates a composite functional system by fusing the engineered mini-Cas14 effector with optimized guide RNAs and delivery vectors. This composite approach integrates the compact protein with complementary molecular components to achieve synergistic effects, resulting in highly efficient genome editing in mammalian cells that neither component could achieve alone.
3Adaptability or versatility
If AAV delivery is used for gene therapy, then therapeutic application is enabled, but the limited payload packaging capacity restricts the size of deliverable genetic material
Solution Approach 1:
The patent extracts the essential coding sequence for the Cas effector function, removing all non-essential sequences and regulatory elements. This extraction creates a minimal genetic payload that fits within the AAV packaging capacity, enabling delivery of the therapeutic gene to mammalian cells while maintaining the full therapeutic effect.
Solution Approach 2:
The patent segments the genome editing function into separate components: a compact Cas14 effector protein, guide RNAs, and PAM sequences in the target DNA. This segmentation allows the Cas effector to be delivered as a small protein or mRNA, while the guide RNAs and target sites provide the specificity, distributing the functional requirements across multiple small elements that collectively fit within AAV capacity limits.
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.