D2S Effector Proteins for Nucleic Acid Editing in Viscous Samples
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Solution Overview
Problem
Existing programmable nucleases face challenges in maintaining specificity and efficiency across a wide range of biological sample conditions, particularly in high viscosity and metal chelating environments.
Innovation Solution
The development of dual-guided short effector proteins (D2S effector proteins) that are compact in size, capable of recognizing various protospacer adjacent motifs (PAMs), and can perform both cis cleavage and trans-collateral cleavage activities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional programmable nucleases are used, then they can cleave nucleic acids in a sequence-specific manner, but they lose accuracy and effectiveness in high viscosity and metal chelating biological sample conditions
Solution Approach 1:
The patent modifies the programmable nuclease system by changing key parameters: using shortened guide RNAs (18-24 nucleotides), incorporating PAM recognition, and optimizing the nuclease structure to tolerate varying environmental conditions. These parameter changes enable the nuclease to maintain specificity and efficiency across diverse biological sample conditions including high viscosity and metal chelating environments.
Solution Approach 2:
The patent introduces PAM (protospacer adjacent motif) recognition as an intermediary mechanism that enhances the nuclease's ability to distinguish target sequences from non-target sequences. This intermediary PAM recognition system provides an additional layer of specificity that maintains accuracy even in challenging biological sample conditions.
2Reliability
If longer effector proteins are used, then they may provide more robust nuclease activity, but they reduce delivery efficiency via viral vectors
Solution Approach 1:
The patent extracts and shortens the effector protein to a minimal functional length (less than 700 amino acids, preferably less than 500 amino acids). This extraction of unnecessary protein length while retaining essential nuclease activity enables efficient delivery via viral vectors while maintaining robust cleavage function.
Solution Approach 2:
The shortened effector protein is designed to perform multiple functions: PAM recognition, target sequence binding, and nuclease activity. This multi-functionality in a compact protein structure maintains robust nuclease activity while improving delivery efficiency through reduced protein size.
3Measurement precision
If standard guide RNAs are used, then they can bind target regions, but they cannot efficiently perform trans-collateral cleavage activity
Solution Approach 1:
The patent employs dynamic guide RNA structures that can adapt to different target configurations. The guide RNA system dynamically switches between cis-cleavage mode (for direct target binding) and trans-collateral cleavage mode (for indirect target modification), enabling both high specificity binding and efficient trans-cleavage activity.
Solution Approach 2:
The patent modifies the guide RNA parameters including length (18-24 nucleotides), sequence composition, and structural features to enable both cis and trans cleavage activities. These parameter changes allow the same guide RNA system to perform both target binding and trans-collateral cleavage efficiently.
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 D2S effector proteins demonstrate enhanced specificity and efficiency in modifying, detecting, and engineering target nucleic acids across diverse sample conditions, with advantages such as reduced spontaneous repair and improved delivery via viral vectors.
Implementation Method 1
Programmable nucleases are proteins that bind and cleave nucleic acids in a sequence-specific manner
Implementation Method 2
guide nucleic acids comprise a CRISPR RNA (crRNA) that is at least partially complementary to a target nucleic acid
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.