In-Partition Probe Barcoding for CRISPR RNA Without Reverse Transcription
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Solution Overview
Problem
Existing methods for analyzing CRISPR-perturbed samples require reverse transcription, which is error-prone and inefficient, and lack efficient methods for processing and analyzing nucleic acids and proteins within partitions like droplets or wells.
Innovation Solution
A method involving hybridization of probes to target regions of nucleic acid molecules, linking probes, and attaching barcode sequences within partitions to generate barcoded nucleic acid molecules, eliminating the need for reverse transcription and enabling controlled analysis of CRISPR-perturbed samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If reverse transcription is used to analyze RNA molecules, then RNA can be converted to DNA for analysis, but the process is error-prone and inefficient
Solution Approach 1:
The invention extracts and eliminates the reverse transcription step from the RNA analysis workflow. By using probe-based methods that directly hybridize to RNA targets and incorporate barcodes without conversion to cDNA, the patent removes the error-prone reverse transcription enzyme step while maintaining the ability to analyze RNA molecules accurately and efficiently
Solution Approach 2:
The invention substitutes the enzymatic reverse transcription mechanism with a hybridization-based detection mechanism. Instead of using reverse transcriptase to convert RNA to cDNA, the patent employs probe hybridization followed by barcode incorporation through polymerase extension, replacing the mechanical/enzymatic conversion process with a more reliable detection approach
2Loss of information
If multiple probes are hybridized to different target regions of the same nucleic acid molecule, then comprehensive analysis of the molecule is achieved, but the complexity of linking and processing multiple probes increases
Solution Approach 1:
The invention merges multiple separate probe molecules into a single linked probe complex. By providing conditions for the probes to link together after hybridizing to different target regions of the same nucleic acid molecule, the patent combines multiple analysis targets into one unified structure that can be processed and barcoded as a single unit, reducing system complexity
Solution Approach 2:
The invention introduces a linking mechanism as an intermediary between multiple hybridized probes. This linker allows probes bound to different target regions to be connected through a bridging structure, enabling the system to handle multiple targets without requiring separate processing pathways for each probe
3Adaptability or versatility
If barcoding is performed within partitions such as droplets or wells, then controlled analysis of individual cells or nucleic acid molecules is enabled, but the precision of barcode attachment to nucleic acid molecules must be maintained
Solution Approach 1:
The invention performs preliminary hybridization of probes to target regions before barcoding within the partition. By ensuring probes are already bound to their specific targets through hybridization before the barcoding step occurs, the patent establishes a stable foundation that maintains precision during the subsequent barcode attachment process within the droplet or well environment
Solution Approach 2:
The invention applies barcoding locally within each partition (droplet or well) rather than in a bulk solution. This localized approach ensures that barcode attachment occurs in a controlled microenvironment where individual nucleic acid molecules or cells can be processed separately, maintaining precision while enabling versatile sample processing
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 accurate and efficient analysis of CRISPR-perturbed samples by generating barcoded nucleic acid molecules without reverse transcription, facilitating sensitive gene expression profiling and multiplexed analysis of nucleic acids and proteins.
Implementation Method 1
hybridizing a probe to a target region of a nucleic acid molecule
Implementation Method 2
performing an extension, denaturation, and amplification processes
Implementation Method 3
performing an extension, denaturation, and amplification processes
Data Source
AI summary
The present disclosure provides methods of processing or analyzing a sample. A method for processing a sample may comprise hybridizing a probe molecule to a target region of a nucleic acid molecule (e.g., a ribonucleic acid (RNA) molecule), barcoding the probe-nucleic acid molecule complex, and performing extension, denaturation, and amplification processes. The nucleic acid molecule may be a nucleic acid molecule associated with CRISPR, (e.g., a guide RNA). A method for processing a sample may comprise hybridizing first and second probes to adjacent or non-adjacent target regions of a nucleic acid molecule (e.g., an RNA molecule such as a guide RNA molecule), linking the first and second probes to provide a probe-linked nucleic acid molecule, and barcoding the probe-linked nucleic acid molecule. One or more processes of the methods described herein may be performed within a partition, such as a droplet or well. One or more processes of the methods described herein may be performed on a cell, such as a permeabilized cell.