CRISPR Enzyme Optimization via Barcoded Oligonucleotide Single-Cell Screening
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Solution Overview
Problem
Current enzymatic activity assays are limited to bulk inputs, requiring large quantities of enzymes and reactants, making them slow and costly, and there is a need for technologies that can accurately measure enzymatic activity at a single cell level for modified enzymes generated through CRISPR gene editing.
Innovation Solution
A reaction mixture comprising a reactant barcoded oligonucleotide (RBO) construct, an amplification construct, and a blocking construct, which allows for the assessment of enzymatic activity by combining these components with an enzyme in a reaction mixture, restricting movement of the amplification construct relative to the RBO construct, and generating a reverse complement of the reaction barcode sequence using an amplification enzyme.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bulk enzymatic activity assays are used, then large quantities of enzymes and reactants can be processed, but the measurement precision and speed are reduced due to the inability to detect single-cell level activity
Solution Approach 1:
The patent segments the bulk enzymatic activity assay into single-cell level measurements by partitioning the reaction mixture into individual compartments (droplets or wells), each containing a single cell. This allows parallel measurement of enzymatic activity across many individual cells, achieving both high measurement precision at single-cell level and efficient processing of large numbers of cells simultaneously.
Solution Approach 2:
The patent uses barcode sequences attached to oligonucleotides as informational copies that can be amplified and detected. Each cell's enzymatic activity is linked to a unique barcode that can be copied through PCR amplification, allowing the activity signal from a single cell to be detected with high precision without requiring large quantities of the original enzymatic reactants.
2Productivity
If bulk enzymatic activity assays are used, then large quantities of enzymes can be processed, but the productivity is reduced due to the slow and costly nature of the process
Solution Approach 1:
By partitioning the assay into single-cell compartments, the system enables parallel processing of thousands of individual cells simultaneously. Each compartment performs the enzymatic reaction and detection independently, allowing high-throughput screening that dramatically increases productivity compared to traditional sequential bulk assays.
Solution Approach 2:
The patent employs self-amplifying barcode sequences that automatically multiply the detection signal through PCR amplification within each compartment. This self-service mechanism allows the system to detect single-cell enzymatic activity without requiring large quantities of external reagents or complex detection equipment, thereby increasing productivity while reducing material consumption.
3Adaptability or versatility
If CRISPR enzyme modification is performed, then customized enzymatic activity can be achieved, but the ability to detect and assess activity at single-cell level is lost
Solution Approach 1:
The patent introduces barcode-containing oligonucleotides as intermediary molecules that link the CRISPR-modified enzyme to the detection system. The barcode acts as a mediator that carries information about the enzymatic activity from the single-cell level to the detection apparatus, enabling the measurement of customized enzyme variants without compromising single-cell detection capability.
Solution Approach 2:
The patent uses amplifiable barcode sequences as informational copies that can be detected through PCR. Each CRISPR-modified enzyme variant is associated with a specific barcode that can be copied and amplified, allowing the detection and assessment of customized enzymatic activity at single-cell level through sequence-based identification rather than direct enzymatic measurement.
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 the detection and assessment of enzymatic activity at a single cell level, facilitating the optimization of enzymatic activity for specific applications by screening large numbers of enzyme variants efficiently and accurately.
Implementation Method 1
generating a reverse complement of the reaction barcode sequence with an amplification enzyme
Implementation Method 2
enzymatically reacting the first reactant with the second reactant to produce a reaction product
Data Source
AI summary
Provided herein are reaction mixtures, compositions, systems, methods, and kits for assessing enzymatic activity. Aspects of the disclosure include use of a reactant barcoded oligonucleotide (RBO) construct comprising a first oligonucleotide comprising a reaction barcode sequence and a first linker that connects the first oligonucleotide to a first reactant, an amplification construct comprising a second oligonucleotide that is complementary to at least a portion of the first oligonucleotide and a second linker that connects the second oligonucleotide to a second reactant, and a blocking construct, comprising a third oligonucleotide that is complementary to at least a portion of the first oligonucleotide. A rate of reaction product generation may be measured to determine an enzymatic activity of a given enzyme.