Molecular information storage using CRISPR
- UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
- Robert F. Glass (Creator)
- Institution
- The University of North Carolina at Greensboro (UNCG )
- Web Site: http://library.uncg.edu/
- Advisor
- Eric Josephs
Abstract: DNA (deoxyribonucleic acids) can be used as a digital data storage medium. Using its nucleotide sequence as a basis for storing digital data, DNA has a significantly higher data density and longer lifespan compared to silicon-based digital data storage technologies. However, the drawbacks of synthesizing long sequences of nucleotides to record data on demand are so significant that they limit the use of DNA for this purpose. This dissertation will discuss methods to circumvent the requirement to synthesize nucleotides and proposes that we instead directly encode digital information onto a standardized double-stranded DNA (dsDNA) template in vitro through targeted mutations. This encoding scheme uses nucleobase (base) editing enzymes to alter DNA sequences at specific sites in pre-synthesized dsDNA templates as a way to store digital information. This document will explore the application of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) base editors to encode digital data into DNA molecules and how we can use DNA sequencing technologies like nanopore sequencing to extract data from these molecules. We have developed an approach to apply CRISPR “base editing” reactions in vitro, where we chemically convert specific nucleotides from one to another onto pre-synthesized DNA “templates” to encode and extract >1250 bits, like they were a magnetic tape, using nanopore sequencing. After developing our in vitro biochemical encoding strategy and optimizing the nucleotide targets to be mutated, we demonstrate that we can direct CRISPR base editing reactions to perform cytosine mutations in vitro using the cytosine deaminase APOBEC3A and a form of the CRISPR enzyme Cas9 (dCas9) that binds to specific 20 bp sequences without cleavage. We then applied our strategy to generate controlled and detectable mutations in vitro and that we can decode our intended data set with 100% accuracy. This document will conclude with future directions to improve our strategy to encode digital data with CRISPR base editing, such as ligating synthesized oligos into unique sequence arrangements and applying alternate deaminating enzymes to generate different mutation patterns. While other scientists have used CRISPR base editing to mutate genomic DNA in cells, this work demonstrates that this technique can be adapted to improve DNA as a medium to store digital data by directly encoding the digital information onto a standardized DNA template via targeted mutagenesis.
Molecular information storage using CRISPR
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Created on 5/1/2023
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Additional Information
- Publication
- Dissertation
- Language: English
- Date: 2023
- Keywords
- Base editing, CRISPR, Data, Deaminase, DNA, Information
- Subjects
- CRISPR-associated protein 9
- Information retrieval