D2S Effector Proteins for Nucleic Acid Editing in Viscous Samples
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Solution Overview
Problem
Programmable nucleases face challenges in accuracy and effectiveness due to challenging biological sample conditions such as high viscosity and metal chelating, limiting their specificity and efficiency across a wide range of sample conditions.
Innovation Solution
Development of dual-guided effector proteins (D2S effector proteins) that are DNA modifying, requiring both crRNA and tracrRNA for activity, and are short in length, leveraging their nucleic acid modifying activities for the modification, detection, and engineering of target nucleic acids, including cis cleavage and trans-collateral cleavage activities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If programmable nucleases are used under challenging biological sample conditions, then nucleic acid cleavage activity is achieved, but accuracy and effectiveness deteriorate due to high viscosity and metal chelating
Solution Approach 1:
The patent employs parameter changes by optimizing the chemical composition of the reaction buffer, including adjusting metal ion concentrations (such as Mg2+ and Ca2+), pH levels, and additive concentrations to counteract the harmful effects of high viscosity and metal chelating in biological samples, thereby maintaining nuclease accuracy and effectiveness
2Ease of operation
If effector proteins are designed to be short in length, then ease of operation and delivery are improved, but structural complexity may be reduced affecting functionality
Solution Approach 1:
The patent extracts and utilizes only the essential functional domains of the effector proteins, removing non-essential regions to create shortened versions that retain core nucleic acid binding and cleavage activities while improving ease of delivery and operation
Solution Approach 2:
The effector proteins are segmented into critical functional modules that can operate independently or in combination, allowing the protein to maintain functionality while being shorter in overall length
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
The D2S effector proteins demonstrate enhanced specificity and efficiency in modifying and detecting target nucleic acids across various sample conditions, improving the accuracy and effectiveness of nucleic acid editing and detection processes.
Implementation Method 1
guide nucleic acids comprise a CRISPR RNA (crRNA) that is at least partially complementary to a target nucleic acid
Implementation Method 2
Programmable nucleases may cleave nucleic acids, including single stranded RNA (ssRNA), double stranded DNA (dsDNA), and single-stranded DNA (ssDNA)
Data Source
AI summary
Provided herein are compositions, systems, and methods comprising effector proteins and uses thereof. These effector proteins are shown to be active with guide RNAs and may be characterized as CRISPR-associated (Cas) proteins. Various compositions, systems, and methods of the present disclosure leverage the activities of these effector proteins for the modification, detection, and engineering of nucleic acids.