Catalytically Inactive MAD7 Nuclease for Sequence-Specific DNA Binding
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Solution Overview
Problem
Current nucleic acid-guided nucleases face challenges in making precise, targeted changes to genomes due to constraints on the range of target sequences they can recognize, which are limited by the need for specific Protospacer Adjacent Motifs (PAMs) near the target sequence.
Innovation Solution
A catalytically inactive MAD7 nuclease variant (dMAD7) derived from Eubacterium rectale, which retains the ability to bind DNA in a sequence-specific manner, allowing for binding to bacterial, yeast, and mammalian genomes without the need for active cleavage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If nucleic acid-guided nucleases are used to make precise, targeted changes to genomes, then gene editing precision is improved, but the range of target sequences is limited by PAM requirements
Solution Approach 1:
The invention extracts the DNA binding function from the nuclease activity by using catalytically inactive nuclease variants (nucleases with mutations that abolish cleavage activity but retain sequence-specific binding). This separation allows the binding function to be used independently for target identification and recruitment without the constraint of requiring active cutting, thereby expanding the range of applicable target sequences while maintaining precise, targeted gene editing capabilities.
2Productivity
If catalytically active nucleases are used for gene editing, then cleavage activity is improved, but the need for specific PAM sequences limits target selection
Solution Approach 1:
The invention extracts the DNA binding function from the nuclease activity by using catalytically inactive nuclease variants (nucleases with mutations that abolish cleavage activity but retain sequence-specific binding). This separation allows the binding function to be used independently for target identification and recruitment without the constraint of requiring active cutting, thereby expanding the range of applicable target sequences while maintaining precise, targeted gene editing capabilities.
Solution Approach 2:
Instead of using active nucleases that require PAM sequences for cleavage, the invention inverts the approach by using inactive nucleases that bind DNA without cutting. This inverted strategy allows the system to function without PAM requirements, fundamentally changing the operational mode from cleavage-dependent to binding-dependent, thereby expanding target sequence versatility.
3Adaptability or versatility
If inactive nuclease variants are used, then PAM requirements are eliminated improving versatility, but cleavage activity is lost
Solution Approach 1:
The invention uses catalytically inactive nuclease variants as intermediaries that bind to target DNA sequences without performing cleavage. These inactive variants serve as mediators to recruit other factors or enable subsequent editing steps without requiring active nuclease activity, thus maintaining versatility while compensating for the loss of direct cleavage function through alternative mechanisms.
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
Enables precise and versatile gene editing by binding to specific DNA sequences, overcoming the limitations of traditional nucleic acid-guided nucleases that require active cutting and specific PAMs, thus enhancing the versatility of gene editing tools.
Implementation Method 1
dMAD7 retains the ability to bind DNA in a sequence-specific manner, allowing for binding to bacterial, yeast, and mammalian genomes
Data Source
AI summary
The present disclosure provides a novel catalytically inactive MAD7 nuclease (dMAD7) that retains the ability to bind DNA in a sequence-specific manner. The MAD7 nuclease from which the dMAD7 has been derived was isolated from Eubacterium rectale.