Genetic circuits for functional screens of Cas12a guide RNA libraries
Author(s)Strait, Elizabeth Ashton.
Program in Media Arts and Sciences (Massachusetts Institute of Technology)
Kevin Michael Esvelt.
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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.
Thesis: S.M., Massachusetts Institute of Technology, School of Architecture and Planning, Program in Media Arts and Sciences, 2019Cataloged from PDF version of thesis.Includes bibliographical references (pages 79-82).
DepartmentProgram in Media Arts and Sciences (Massachusetts Institute of Technology)
Massachusetts Institute of Technology
Program in Media Arts and Sciences