2026-09-20T04:36:37Zhttps://dspace.mit.edu/server/oai/requestoai:dspace.mit.edu:1721.1/1240762021-07-05T14:03:20Zcom_1721.1_7582com_1721.1_7581col_1721.1_131023
Kevin Michael Esvelt.
Strait, Elizabeth Ashton.
Program in Media Arts and Sciences (Massachusetts Institute of Technology)
Program in Media Arts and Sciences (Massachusetts Institute of Technology)
2020-03-09T18:52:28Z
2020-03-09T18:52:28Z
2019
2019
https://hdl.handle.net/1721.1/124076
1142190545
Thesis: S.M., Massachusetts Institute of Technology, School of Architecture and Planning, Program in Media Arts and Sciences, 2019
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 79-82).
The discovery of CRISPR RNA-guided endonucleases have catalyzed huge technological advancements in the field of synthetic biology, such as the creation of gene drives: genomically encoded CRISPR systems capable of spreading through a wild population. These systems have two components: a CRISPR-associated (Cas) protein and a guide RNA consisting of a conserved "scaffold" sequence recognized by the protein and a variable "spacer" complementary to the DNA target of interest. CRISPR-based gene drives are greatly improved by targeting many sites simultaneously using multiplexed guide arrays; however, due to the conserved scaffold sequence, such arrays introduce significant stretches of homologous repeats that can affect the generational stability of the drive system. Here, I describe the design and use of CRISPR-based gene circuits for screening large libraries of gRNA scaffold variants. These circuits report on the activity of scaffolds for DNA target binding and gRNA processing, a crucial function for multiplexing. The circuits employ prokaryotic transcriptional logic gates and a novel post-transcriptional regulation mechanism to produce fluorescent outputs, which enable FACS sorting of cell libraries with scaffold permutations. Subsequent deep-sequencing of these sorted pools reveals enrichment for a diverse set of highly active, novel functional scaffold sequences. These variants hugely expand the toolbox of Cas12a components available to synthetic biologists, eliminating many of the current barriers to large-scale multiplexing.
by Elizabeth Ashton Strait.
S.M.
S.M. Massachusetts Institute of Technology, School of Architecture and Planning, Program in Media Arts and Sciences
82 pages
eng
Massachusetts Institute of Technology
MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission.
http://dspace.mit.edu/handle/1721.1/7582
Program in Media Arts and Sciences
Genetic circuits for functional screens of Cas12a guide RNA libraries
Thesis
application/pdf
2020-03-09T18:52:27Z
Publication
Master
Media
unknown
unknown
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