Type V dCRISPR Repressors for Specific Gene Silencing
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Solution Overview
Problem
Existing gene silencing methods, such as RNAi and earlier CRISPR/Cas9 systems, face limitations like off-target effects and incomplete knockdown, necessitating the development of improved gene repressor systems for therapeutic and research applications.
Innovation Solution
Compositions and methods utilizing catalytically-dead Class 2 Type V CRISPR proteins (dXR) linked with transcription repressor domains and guide RNAs (gRNA) for targeted gene repression, including vectors and lipid nanoparticles for cellular delivery.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If RNAi is used for targeted-gene knockdown, then gene silencing can be achieved, but off-target effects and incomplete knockdown occur
Solution Approach 1:
The patent changes the molecular mechanism parameter from RNAi-based post-transcriptional silencing to CRISPR/dCas9-based transcriptional repression. This parameter change enables specific gene silencing through programmable DNA binding, eliminating off-target effects while achieving complete knockdown through direct transcriptional blocking.
Solution Approach 2:
The patent replaces the RNAi mechanical system (small interfering RNA binding to target mRNA) with a protein-based CRISPR system (dCas9-gRNA complex binding to target DNA). This substitution provides more precise target recognition and complete transcriptional repression without the off-target effects inherent in RNAi.
2Reliability
If tailored DNA binding proteins such as zinc finger proteins or TALEs are used, then selective gene suppression can be achieved, but each desired target gene necessitates the generation of a new protein
Solution Approach 1:
The patent creates a universal dCas9 platform that can target any gene through programmable gRNA sequences. Instead of generating new proteins for each target, the same dCas9 protein works with different gRNAs to achieve selective gene suppression across multiple targets, dramatically simplifying the system.
Solution Approach 2:
The patent uses gRNA as a programmable copy or template that directs the universal dCas9 protein to specific target sequences. Rather than creating new proteins for each target, the system copies the targeting information into RNA sequences that guide the reusable dCas9 protein to the desired genomic locations.
3Adaptability or versatility
If CRISPR/Cas9 systems are used for gene editing, then genomic manipulation capability is achieved, but gene silencing without editing is not optimal for certain disease indications
Solution Approach 1:
The patent extracts the DNA binding and transcriptional repression functions from the Cas9 nuclease and separates them from the gene editing function. By using catalytically inactive dCas9, the system retains genomic targeting and transcriptional regulation capabilities while eliminating the DNA-cutting activity, making it appropriate for gene silencing applications where editing is not desired.
Data Source
AI summary
The disclosure relates to gene repressor systems comprising catalytically-dead Class 2 CRISPR proteins and one or more transcription repressor domains linked to the catalytically-dead Class 2 CRISPR protein as a fusion protein, as well as a guide ribonucleic acid (gRNA); and methods of making and using same.