Hypercascade Arrays for Lineage Reconstruction

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Solution Overview

Problem

Existing molecular recording systems for cells have limited information capacity and disrupt spatial context, making it difficult to reconstruct the dynamic histories of individual cells and their lineage relationships over long time scales.

Innovation Solution

The system comprises hypercascade arrays with multiple hypercascade units, layer guide RNAs, and a base editor capable of adenine-to-guanine or cytosine-to-thymine base editing, allowing for extended editing durations and deep lineage reconstruction.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Loss of information

If existing molecular recording systems are used to capture cell lineage relationships, then lineage information can be recorded, but the information capacity is limited and spatial context is disrupted

Engineering Contradiction:

Improvelineage information capacityVSAvoidspatial context disruption

Core Design Contradiction:

Loss of informationVSDevice complexity

Solution Approach 1:

The system segments the recording function into multiple independent hypercascade units (HCUs), each containing target sites that can be edited by specific guide RNAs. This segmentation allows parallel recording of multiple lineage events without interfering with spatial context, as each unit operates independently within the genomic locus.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The system implements nested recording layers where outer hypercascade units are edited first, then inner units become accessible for subsequent editing. This nested structure enables hierarchical recording of lineage relationships over multiple generations while maintaining a compact genomic footprint that preserves spatial context.

Inventive Principle:

Principle #7Nested doll (Nesting)

2Duration of action of stationary object

If existing recording systems operate over long time scales, then deep lineage reconstruction is attempted, but barcode loss becomes non-linear and accuracy decreases

Engineering Contradiction:

Improverecording durationVSAvoidlineage reconstruction accuracy

Core Design Contradiction:

Duration of action of stationary objectVSMeasurement precision

Solution Approach 1:

The system ensures continuous useful action by designing hypercascade units with conditional target sites that become editable only after specific preceding edits occur. This continuous cascade of editable targets ensures that lineage recording remains accurate over extended periods, as each generation's edits enable the next generation's recording capacity.

Inventive Principle:

Principle #20Continuity of useful action

Solution Approach 2:

The system dynamically activates different sets of target sites based on the current editing state. As outer hypercascade units are edited, inner units transition from inaccessible to editable states, allowing the system to adapt its recording capacity over time and maintain precision across multiple generations.

Inventive Principle:

Principle #15Dynamics

3Loss of information

If multiple target sites are edited simultaneously, then information capacity increases, but mismatch repair prevents accurate recording of conditional targets

Engineering Contradiction:

Improveinformation capacityVSAvoidconditional target editing accuracy

Core Design Contradiction:

Loss of informationVSReliability

Solution Approach 1:

The system performs preliminary editing of outer hypercascade units before making inner units editable. This preliminary action creates the necessary conditions (repair of mismatches in guide RNA binding sites) that enable subsequent accurate editing of conditional target sites, ensuring both high information capacity and reliability.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The system cushions against editing errors by designing conditional target sites with intentional mismatches that prevent premature or inaccurate editing. These mismatches act as protective barriers that only become removable after specific preceding edits occur, ensuring that conditional targets are edited only under the correct biological conditions.

Inventive Principle:

Principle #11Beforehand cushioning (Prior cushioning)

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

The system achieves near-linear barcode loss over time, enabling accurate lineage reconstruction for multiple generations and maintaining spatial context, thus overcoming the limitations of existing technologies.

Implementation Method 1

an editor, wherein the editor is a base editor capable of base editing thehypercascade array. In some embodiments, said base editing comprises: adenine (A)-to-guanine (G) base editing and/or cytosine (C)-to-thymine (T) base editing

Methodology Applied

Scientific EffectBase editing:

Data Source

PatentUS20250075202A1Molecular recording methods and systems to capture lineage relationships in differentiating stem cells

Publication Date: 2025.03.06 CALIFORNIA INST OF TECH

AI summary

Disclosed herein include methods, compositions, and kits suitable for use in capturing lineage relationships in dividing cell populations. Disclosed herein include compact phylogenetic recording systems for high resolution lineage reconstruction over long time scales. In some embodiments, the system comprises one or more hypercascade array(s) each comprising p hypercascade units, n layer guide RNAs, and an editor. In some embodiments, the system comprises one or more target array(s) comprising n editable target sites, n guide RNAs gRNAs, and a base editor capable of adenine (A)-to-guanine (G) base editing.