MAD Nucleases with Altered PAM Specificity for Genome Editing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current nucleic acid-guided nucleases have limited versatility due to constrained target sequence recognition, requiring specific PAMs near the target sequence, which restricts precise and targeted genome editing in living cells.
Innovation Solution
Development of Type V MAD nucleases with varied PAM preferences and activities, allowing for broader recognition of target sequences and increased editing capabilities in mammalian cells, including the MAD293, MAD294, MAD295, MAD296, MAD297, MAD298, and MAD299 systems.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If nucleic acid-guided nucleases are used for genome editing, then precise targeted changes can be made to the genome, but the range of target sequences is constrained by specific PAM requirements
Solution Approach 1:
The patent applies parameter changes by modifying the PAM recognition requirements of nucleic acid-guided nucleases. By engineering nucleases with altered PAM specificities (e.g., changing from NGG to other sequences), the patent expands the range of targetable genomic sequences while maintaining precise editing capability at each target site.
Solution Approach 2:
The patent develops multiple nuclease variants (e.g., SpCas9 variants, SaCas9 variants, and other Type V nucleases) with different PAM preferences. This creates a universal genome editing toolkit where different nucleases can be selected based on the specific genomic context, enabling broad applicability across diverse target sequences while preserving precise editing function.
2Manufacturing precision
If existing nucleases with specific PAM requirements are used, then editing can be performed at targeted locations, but versatility for different editing tasks is limited
Solution Approach 1:
The patent systematically varies PAM sequence parameters across different nuclease variants to match different genomic contexts. By tuning PAM recognition stringency and sequence preferences, the patent enables versatile application across diverse editing tasks including knockouts, knockins, and base editing while maintaining precise targeted editing at each specific locus.
Solution Approach 2:
The patent introduces dynamic selection of nuclease variants based on the specific editing task and genomic target. Different nucleases with varying PAM specificities can be dynamically chosen depending on the required precision, target location, and editing type, creating a flexible and adaptable genome editing platform.
Data Source
AI summary
The present disclosure provides new RNA-guided nucleases for making rational, direct edits to nucleic acids in live cells; specifically, the present disclosure provides Type V MAD nucleases (e.g., RNA-guided nucleases or RGNs) with altered PAM preferences and/or altered activity at different temperatures or fidelity, and/or varied nuclease activities; all changes that may increase the versatility of a nucleic acid-guided nuclease for certain editing tasks.