Engineered Gene Effectors for Compact CRISPR Gene Regulation
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Solution Overview
Problem
Existing gene regulation technologies fail to effectively address the challenges of optimizing gene expression and activity regulation, as benchmark effectors like VPR and KRAB are either too large for efficient packaging or insufficient for comprehensive gene regulation.
Innovation Solution
Development of engineered gene effectors with sizes less than or equal to 500 amino acids, comprising at most 70% sequence identity to SEQ ID NO: 1, and comprising at least a portion of VP16, VP64, p65, and Rta; the engineered gene effector has a size less than or equal to about 250 amino acids, capable of activating or repressing a target gene in a cell.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the VPR (VP64-p65-Rta fusion polypeptide) is used as a gene effector, then the gene activation capability is strong, but the size (about 518 amino acid residues) is too large for efficient packaging in viral vectors
Solution Approach 1:
The patent divides the VPR effector into smaller functional modules by selecting and combining specific transcriptional activation domains (VP16, VP64, p65, or Rta) rather than using the full-length VPR. This segmentation allows the effector to maintain strong gene activation capability while reducing the overall size to 250-500 amino acids for efficient viral vector packaging.
Solution Approach 2:
The patent extracts the essential transcriptional activation functions from the full VPR effector by identifying and isolating specific functional domains (VP16, VP64, p65, Rta). These extracted domains are then recombined into mini-effectors that retain the necessary gene activation capability without the bulk of the original VPR structure.
2Reliability
If the KRAB domain is used as a gene effector, then the gene repression capability is achieved, but the effector may not be optimal or sufficient for regulating all genes
Solution Approach 1:
The patent creates multi-functional effectors by combining different transcriptional activation domains (VP16, VP64, p65, Rta) that can regulate different sets of genes. This combination allows a single effector construct to potentially regulate multiple gene sets with different activation mechanisms, thereby expanding regulatory coverage and adaptability while maintaining repression capability through the KRAB domain.
3Ease of manufacture
If the engineered gene effector size is reduced to at most 500 amino acid residues, then the packaging efficiency in viral vectors is improved, but the regulatory capability may be compromised
Solution Approach 1:
The patent optimizes the effector size parameter by carefully selecting and combining transcriptional activation domains to achieve a balanced construct size (250-500 amino acids). This parameter optimization ensures the effector is small enough for efficient viral vector packaging while maintaining sufficient regulatory capability through the strategic selection of high-activity domains.
Data Source
AI summary
The present disclosure provides one or more engineered gene effectors and systems, compositions, and methods of use thereof, wherein the one or more engineered gene effectors can be used to effect regulation of a target gene in a cell (e.g., an endogenous target gene in a cell). The one or more engineered gene effectors can be operatively coupled to a heterologous endonuclease, such as a CRISPR/Cas protein.