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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

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A time-resolved, multi-symbol molecular recorder via sequential genome editing.

Junhong Choi^1,2, Wei Chen^3,4, Anna Minkina³

¹Department of Genome Sciences, University of Washington, Seattle, WA, USA. junhongc@uw.edu.

|July 6, 2022

Summary

DNA Typewriter enables precise in vivo molecular recording by using sequential genome editing to capture event order and thousands of symbols. This DNA-based system overcomes limitations of current recording technologies.

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Area of Science:

Molecular Biology
Genetics
Biotechnology

Background:

DNA is a suitable medium for in vivo molecular recording.
Current DNA-based memory devices have limitations in recording capacity and event order.
Existing technologies struggle to capture complex biological histories.

Purpose of the Study:

To introduce DNA Typewriter, a novel system for in vivo molecular recording.
To overcome limitations of existing DNA-based recording methods.
To demonstrate high-capacity, order-preserving recording of biological events.

Main Methods:

Developed a 'DNA Tape' using tandem CRISPR-Cas9 target sites.
Utilized short insertional edits as symbols to record prime editing guide RNA identity.
Implemented sequential genome editing for order-preserving recording.
Integrated with single-cell RNA sequencing for lineage reconstruction.

Main Results:

Demonstrated recording and decoding of thousands of symbols and complex event histories.
Successfully recorded short text messages.
Evaluated performance of orthogonal DNA tapes and constructed 'long tapes' for serial events.
Reconstructed a monophyletic lineage of 3,257 cells.
Showed that sequential edits are maintained across at least 20 generations and 25 days.

Conclusions:

DNA Typewriter offers a robust platform for high-capacity, order-preserving in vivo molecular recording.
The system successfully captures complex biological event histories and cellular lineages.
This technology advances the potential of DNA for digital data storage and biological recording.