Genetic circuits for functional screens of Cas12a guide RNA libraries
Name
1142190545-MIT.pdf
Size
25.31 MB
Format
Adobe PDF
Checksum (MD5)
69dd05b7ddf4331f3e5eb2ba15760d68
Author(s)
Strait, Elizabeth Ashton.
Advisor(s)
Kevin Michael Esvelt.
Date Issued
2019
Publisher
Massachusetts Institute of Technology
Abstract
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.
Description
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).
Subjects
Program in Media Arts and Sciences
MIT Department
Program in Media Arts and Sciences (Massachusetts Institute of Technology)
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