{"id":{"repo_id":"mit","oai_identifier":"oai:dspace.mit.edu:1721.1/124076"},"canonical_url":"https://search.dev.ndltd.org/etd/mit/oai:dspace.mit.edu:1721.1/124076","repository":{"repo_id":"mit","name":"MIT","base_url":"https://dspace.mit.edu/oai/request"},"display":{"title":"Genetic circuits for functional screens of Cas12a guide RNA libraries","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.","abstract_html":"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.","abstract_has_math":false,"creators":["Strait, Elizabeth Ashton."],"institution":"Massachusetts Institute of Technology","degree_name":"Master","degree_level":null,"degree_discipline":null,"degree_department":"Program in Media Arts and Sciences (Massachusetts Institute of Technology)","school":null,"contributors":[],"advisors":["Kevin Michael Esvelt."],"committee_chairs":[],"committee_members":[],"year":2019,"date_issued":"2019","date_published":"2019","updated_at":"2026-07-22T22:22:26Z","subjects":["Program in Media Arts and Sciences"],"languages":["eng"],"rights":["MIT theses are protected by copyright. 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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. 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