Effector Proteins for Nucleic Acid Editing in Challenging Conditions
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Solution Overview
Problem
Programmable nucleases face challenges in accuracy and effectiveness across various biological and sample conditions, particularly in high viscosity and metal chelating environments, limiting their sequence-specific editing and detection capabilities.
Innovation Solution
Compositions and systems comprising effector proteins and guide nucleic acids with specific amino acid sequences and nucleotide sequences that are at least 75% to 100% identical to described sequences, enabling efficient cis and trans cleavage activities, and modified to retain sequence selectivity while reducing nuclease activity, are used for nucleic acid modification and detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If programmable nucleases are used for sequence-specific nucleic acid editing and detection, then editing and detection capabilities are achieved, but accuracy and effectiveness are limited under challenging biological and sample conditions such as high viscosity and metal chelating environments
Solution Approach 1:
The patent modifies the programmable nuclease by changing amino acid parameters at specific positions (e.g., substituting residues involved in metal ion coordination) to alter its biochemical properties. This allows the enzyme to maintain catalytic activity in metal-chelating conditions where wild-type nucleases fail, directly addressing the adaptability issue while preserving editing accuracy
Solution Approach 2:
The patent introduces modified guide RNA molecules as intermediaries that facilitate the interaction between the engineered nuclease and target nucleic acids under challenging conditions. These guide RNAs are designed with enhanced stability and binding affinity, serving as mediators that protect the nuclease complex from the adverse effects of high viscosity and metal chelation, thereby improving both reliability and adaptability
2Quantity of substance
If amino acid residues that impart catalytic activity are substituted to reduce nuclease activity, then nuclease activity is reduced, but sequence selectivity must be retained
Solution Approach 1:
The patent applies local quality by making site-specific amino acid substitutions only at positions responsible for catalytic activity (such as residues in the catalytic pocket that coordinate metal ions), while leaving unchanged the amino acid residues that form the sequence-specific binding interface. This localized modification reduces overall nuclease activity while preserving sequence selectivity, as the binding recognition regions remain intact
Solution Approach 2:
The patent segments the functional analysis of the nuclease into distinct regions: the catalytic core (where substitutions are made to reduce activity) and the guide RNA binding/sequence recognition region (where substitutions are avoided to maintain selectivity). This functional segmentation allows independent optimization of activity level and specificity, resolving the contradiction between reducing quantity of substance and maintaining manufacturing precision
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
These compositions and systems enhance the specificity and efficiency of nucleic acid editing and detection across a wide range of conditions, improving the accuracy and reliability of programmable nuclease applications.
Implementation Method 1
a guide nucleic acid that imparts activity or sequence selectivity to the programmable nuclease
Implementation Method 2
Programmable nucleases are proteins that bind and cleave nucleic acids in a sequence-specific manner
Data Source
AI summary
Provided herein are compositions, systems, and methods comprising effector proteins and uses thereof. These effector proteins may be characterized as CRISPR-associated (Cas) proteins. Various compositions, systems, and methods of the present disclosure may leverage the activities of these effector proteins for the modification, detection, and engineering of nucleic acids.