Nucleic Acid-Modifying Enzyme Assays with Compartmentalized Sequencing
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Solution Overview
Problem
Current methods for engineering nucleic acid modifying enzymes like CRISPR-Cas are limited by the inability to efficiently screen and identify functional variants from large libraries, lacking scalability and precision in measuring enzymatic activity.
Innovation Solution
A method involving compartmentalization of polynucleotide constructs in individual compartments for in vitro expression, followed by single molecule sequencing to detect and count modified and unmodified DNA/RNA molecules, enabling high-throughput screening of enzyme variants and their targets.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If selection-based approaches are used to engineer nucleic acid modifying enzymes, then active variants can be identified through enrichment, but the degree of protein activity cannot be measured and inactive variants provide no information
Solution Approach 1:
The patent segments the enzyme library into individual compartments (droplets or wells), each containing a single enzyme variant and its encoding DNA. This segmentation enables independent measurement of each variant's activity through single-molecule sequencing, allowing precise quantification of activity levels for both active and inactive variants without information loss.
2Measurement precision
If screening approaches are used to measure enzyme activity, then both active and inactive variants can be measured, but the method is not scalable due to increased resources needed
Solution Approach 1:
The patent replaces traditional mechanical/chemical detection methods with single-molecule sequencing technology. By using sequencing to detect and count modified versus unmodified DNA molecules, the system achieves high-throughput screening capability that scales efficiently, measuring thousands of variants simultaneously without the resource constraints of conventional screening methods.
3Measurement precision
If compartmentalization is implemented for single-molecule sequencing, then direct measurement of enzymatic activity is enabled, but the device complexity increases
Solution Approach 1:
The patent employs a universal polynucleotide construct design that can encode any nucleic acid modifying enzyme variant and its target sequence. This multi-functional construct system works across different enzyme types and applications, reducing the need for separate specialized systems and thereby managing complexity while maintaining measurement 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
Enables direct measurement of enzymatic activity and inactivity for each variant, allowing for enhanced engineering of nucleic acid modifying enzymes and optimization of guide RNAs and targets, with scalability for large libraries.
Implementation Method 1
subjecting the plurality of the compartments to conditions which allow the modification of DNA/RNA targets by nucleic acid modifying enzymes which have modification activity towards said DNA or RNA targets
Data Source
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AI summary
The present application discloses a multiplex method to measure the activities of nucleic acid modifying enzymes and screen one or more variable elements of the enzymatic reaction. The method comprises: (i) providing a library of polynucleotide constructs encoding a DNA/RNA target site and a variable element to be tested e.g. a nucleic acid modifying enzyme variant, (ii) compartmentalizing single copies of the polynucleotide constructs together with in vitro transcription translation (IVVT) reagents, (iii) allowing IVVT reactions that express the nucleic acid modifying enzyme and DNA/RNA target site in each compartment, (iv) individual polynucleotide construct/RNA target will be cleaved, intact, or otherwise modified depending on the functionality of the encoded nucleic acid modifying enzyme, and (v) quantifying the cleaved, intact, or modified polynucleotide construct/RNA target by sequencing, thereby directly identifying and quantifying the enzymatic activity associated with each variable element. In particular, the nucleic acid modifying enzyme is a CRISPR-associated protein (Cas).