CRISPRa Activation of Complex Genomic Loci
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Solution Overview
Problem
Current methods for overexpressing complex genomic loci are limited by the need to identify and overexpress individual components, which is challenging due to multiple promoters and incomplete annotation of transcripts, especially for large transcripts and complex loci.
Innovation Solution
The method involves using CRISPRa technology to activate the main regulatory element of a complex genomic locus, such as a promoter, to globally enhance the transcription of an entire transcriptional set, thereby overcoming the need to identify individual components.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If individual transcripts are overexpressed using plasmids or viral vectors, then specific transcript function can be studied, but the method becomes infeasible when transcript size exceeds vector limits or when multiple transcripts need simultaneous overexpression
Solution Approach 1:
The patent merges multiple individual transcript overexpression strategies into a single integrated approach by targeting the common promoter region of a complex genomic locus. This allows simultaneous overexpression of multiple transcripts (including lncRNAs and microRNAs) through one intervention, eliminating the need for separate vectors for each component and overcoming vector size limitations.
Solution Approach 2:
The patent identifies a universal regulatory mechanism (common promoter region) that controls multiple transcripts within a complex locus. By targeting this universal element, the method achieves multi-functional overexpression of different transcript types (coding and noncoding RNAs) simultaneously, making the approach applicable to various transcript combinations without requiring separate strategies for each.
2Measurement precision
If multiple promoters within a complex locus are targeted individually, then specific transcripts can be activated, but the difficulty of identifying correct components increases due to incomplete annotation
Solution Approach 1:
The patent extracts the common promoter region as a key regulatory element that is shared across multiple transcripts within a complex locus. By focusing on this extracted common element rather than attempting to target each individual promoter separately, the method simplifies the identification process and enables accurate activation of multiple transcripts simultaneously, overcoming the complexity introduced by multiple promoters and incomplete annotation.
3Reliability
If individual transcripts are overexpressed, then therapeutic modulation of specific pathways is achieved, but the approach cannot restore physiological function when multiple transcripts from the same locus are required
Solution Approach 1:
The patent combines the overexpression of multiple transcripts (lncRNAs and microRNAs) that are normally co-regulated by a common promoter into a single therapeutic intervention. This merging approach restores physiological cell function more effectively than individual overexpression, as it simultaneously re-establishes the coordinated expression of multiple components required for normal cellular processes, thereby improving therapeutic reliability and adaptability.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach allows for the simultaneous activation of multiple transcripts within a complex genomic locus, achieving global transcriptional enhancement and restoring physiological cell function, as demonstrated by activating the CARMN and MIR503HG loci.
Implementation Method 1
The method involves using CRISPRa technology to activate the main regulatory element of a complex genomic locus, such as a promoter, to globally enhance the transcription of an entire transcriptional set
Data Source
AI summary
Disclosed is a construct for use in a method of transcriptional activation of a complex genomic locus. The construct comprises a single guide RNA (sgRNA) binding a regulatory element of the said complex genomic locus, and a deactivated Cas. The construct may be introduced into cells including the genomic locus of interest in order to activate the regulatory element to transcribe multiple transcripts within said complex genomic locus.