Massively-parallel characterization of CRISPR nuclease binding and applications in the creation of CRISPR-based gene circuits

Abstract

Due to their simple and programmable nature, the nucleases of class 2 CRISPR systems have been the subject of intense research interest for the purposes of genome editing, programmable gene regulation, and nucleic acid detection. In this dissertation I exploit freedom in both in the CRISPR identification region as well as bacterial promoter design to multiplex sets of synthetic NOT gate elements in E. coli, each of which is produced using a specific CRISPR guide:target pairing. I use this method to study mismatch tolerance in CRISPR systems and probe their properties, including base binding preferences, position-dependent mismatch tolerance, and PAM site sequence. While initially only applied to FnCas12a, I then utilize this technique for other Cas proteins, including species variants of Cas9, Cas12a, Cas13a, and CasX. These ‘CRISPRgates’ are simple NOT gate elements which can target genes or other CRISPRgate elements and in principle can be combined to create complex synthetic genetic circuits, a fundamental goal of synthetic biology. Repression with CRISPR is advantageous because in principle we can repress many different targets simultaneously without crosstalk. However, such gene circuit elements behave poorly when placed in series due to signal loss that occurs due to leaky repression and retroactive effects owing to a shared pool of Cas proteins. By utilizing antisense RNAs to sequester guide RNA transcripts, I demonstrate a mechanism to suppress leaky CRISPRi repression and restore logical gene circuit function when elements are used in series.