Increasing CRISPR-mediated digital data storage in DNA using engineered “Janus” protospacers
- UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
- Michelle Gonzalez (Creator)
- Institution
- The University of North Carolina at Greensboro (UNCG )
- Web Site: http://library.uncg.edu/
- Advisor
- Eric Josephs
Abstract: DNA molecules can be used to store digital data if the sequence of their nucleotides is assigned to different values of ‘0’ or ‘1’. Rather than having to synthesize new DNA molecules for data storage on demand, our laboratory and its collaborators have developed a method to “overwrite” the nucleotide sequences on a double-stranded DNA molecule by mutating them using CRISPR base editors in vitro so that new information can be stored on these existing DNA molecules. In this system, nucleotides on one strand in the sequences that are recognized by CRISPR effectors (the “protospacers”) are changed from ‘0’ (unmutated) to ‘1’ (mutated). For this thesis, we sought to increase the data storage capacity in this system by engineering protospacer sequences where the CRISPR effectors could effectively recognize sequences on both of the strands (“Janus” protospacers) which is not generally or necessarily the case for a given protospacer sequence. We designed and experimentally validated several Janus protospacer sequences so that, for each of the protospacer sequences, it can potentially store a ‘0’ (unmutated), a ‘1’ (top strand mutated), a ‘2’ (bottom strand mutated), or a ‘3’ (both strands mutated). We were able to engineer Janus protospacers with sequence motifs that should be recognized by mutagens APOBEC3A or APOBEC3G, which have distinct mutational signatures and would further increase the capacity of the systems. In our work here, we attempt to use Janus protospacers so they could effectively double the information storage capacity of DNA molecules used for CRISPR-mediated DNA data storage, at minimum.
Increasing CRISPR-mediated digital data storage in DNA using engineered “Janus” protospacers
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Created on 5/1/2023
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Additional Information
- Publication
- Thesis
- Language: English
- Date: 2023
- Keywords
- Adenine Base Editing, CRISPR-Cas9, Cytosine Base Editing, Nucleic Acid Memory Storage
- Subjects
- DNA nanotechnology
- Nucleotide sequence
- CRISPR-associated protein 9