DNA-Synthesis Barcoding for Single-Cell Connectomics
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Solution Overview
Problem
Current techniques for single cell connectomics lack single-cell resolution due to physical limitations of optical or electrical probes and limited recording characteristics of DNA-based devices, hindering the study of densely packed cell structures like those in the brain.
Innovation Solution
A DNA-synthesis based recording system combining CRISPR-Cas9 or other CRISPR systems with homing guide RNA and terminal deoxynucleotidyl transferase (TdT), where TdT adds nucleotides to DNA breaks, creating a barcode-dependent output signal detectable by in situ sequencing, allowing for single-cell level connectivity mapping.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If optical or electrical probes are used for lineage tracing and connectomics, then recording capability is achieved, but single-cell resolution is lost due to physical limitations
Solution Approach 1:
The patent replaces optical and electrical probe systems with a DNA-based molecular recording system. The CRISPR-Cas9 system creates site-specific DNA breaks, and TdT incorporates nucleotides at these breaks to create permanent genetic records. This substitution of mechanical/optical systems with molecular biological systems enables single-cell resolution while maintaining reliable recording capability through genetically encoded memory.
2Measurement precision
If DNA-based recording devices are used, then single-cell resolution is achieved, but recording characteristics are limited
Solution Approach 1:
The patent changes the parameters of DNA-based recording by incorporating variable nucleotide sequences (barcodes) at CRISPR break sites. The TdT enzyme adds nucleotides with specific sequences that encode information about the timing and nature of cellular events. This parameter change from simple presence/absence recording to sequence-encoded recording dramatically expands the information capacity while maintaining single-cell resolution.
Solution Approach 2:
The patent introduces TdT as an intermediary enzyme that bridges the CRISPR-Cas9 DNA breakage system and the information recording function. TdT incorporates nucleotides at the break sites, acting as a mediator that converts the physical DNA break event into a coded genetic record. This intermediary enables rich information encoding while preserving the single-cell resolution capability of the DNA-based system.
3Loss of information
If barcoding systems are used for connectomics, then connectivity information can be tracked, but single-cell level resolution is not achieved in densely packed cells
Solution Approach 1:
The patent segments the barcoding system to operate at the individual cell level within densely packed tissues. Each cell receives unique CRISPR edits at specific genomic loci, creating cell-specific barcode sequences. The in situ sequencing method then reads these barcodes with single-cell spatial resolution, enabling connectivity tracking while maintaining the ability to distinguish individual cells even in densely packed environments like the brain.
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 single-cell level connectivity mapping in densely packed cell environments, providing high-resolution spatial reconstruction of cells and tissues, including the brain, by synthesizing a DNA-based record responsive to nucleotide concentrations, overcoming previous limitations in resolution and recording capabilities.
Implementation Method 1
A DNA-synthesis based recording system combining CRISPR-Cas9 or other CRISPR systems with homing guide RNA and terminal deoxynucleotidyl transferase (TdT), where TdT adds nucleotides to DNA breaks
Implementation Method 2
the TdT is directed to the double-stranded breaks created by Cas at the hgRNA sites, and the TdT adds at least one nucleotide at the double-stranded breaks
Data Source
AI summary
The present disclosure provides a DNA-synthesis based recording system that, in combination with CRISPR-Cas9 or other CRISPR systems, can establish single-cell level connectivity for densely packed cells, for example in the brain.