Multi-Partite CRISPR Editor Screening for Graded Gene Control
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Solution Overview
Problem
Genome editing technologies face risks of genotoxicity from double-strand breaks and lack of graded gene expression control, necessitating the development of novel epigenomic editing proteins.
Innovation Solution
A method for identifying candidate polypeptides with editing activity by expressing a library of polypeptides with specific domains in cells, separating cells based on target expression levels, and sequencing genomic DNA to determine editing activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If genome editing is performed using nuclease-based methods, then gene editing capability is achieved, but genotoxicity risk increases due to double-strand breaks
Solution Approach 1:
The patent extracts and removes the nuclease domain from the CRISPR-Cas system, retaining only the DNA-binding capability of dCas9. This extraction eliminates the harmful DNA-cleaving function while preserving the targeted DNA binding ability, thereby resolving the contradiction between editing capability and genotoxicity risk
Solution Approach 2:
The patent introduces epigenetic modification domains (such as methyltransferases, demethylases, acetyltransferases, or deacetylases) as intermediary effectors that modify chromatin structure and gene expression without causing DNA breaks. These intermediary proteins mediate the desired gene regulation function while avoiding direct DNA cleavage, thus eliminating genotoxicity
2Manufacturing precision
If nuclease-based genome editing is used, then complete gene knockout is achieved, but graded gene expression control is lost
Solution Approach 1:
The patent changes the mechanism of action from binary (cut/not cut) to continuous by employing epigenetic modifiers that can adjust gene expression levels. Different epigenetic domains (methyltransferases, demethylases, acetyltransferases, deacetylases) and their combinations allow for graded modulation of transcriptional activity, enabling precise control over gene expression levels rather than complete knockout
Solution Approach 2:
The patent creates a universal platform where the same dCas9 scaffold can be fused to multiple different epigenetic effectors to achieve diverse outcomes. By swapping effector domains, the system can produce activation, repression, or graded expression effects, providing both completeness and adaptability in gene regulation
Data Source
AI summary
Provided are methods for determining that a candidate polypeptide in a library of polypeptides has editing activity comprising expressing in a plurality of cells: a library of first expression cassettes each comprising a nucleic acid encoding a polypeptide wherein each polypeptide in the library comprises, a first editing domain, a first linker, a nuclease-deficient RNA guided endonuclease domain, a second linker, and a second editing domain; and a second expression cassette comprising a nucleic acid encoding a guide RNA directed to a target of interest, wherein said first and second expression cassettes are expressed in the plurality of cells; separating the plurality of cells based on the expression level of the target of interest; extracting genomic DNA (gDNA) from the plurality of cells; and sequencing the gDNA of cells that have a high level expression or a low level of expression of the target of interest.