Investigating catalytically inactive Cas proteins as transcriptional roadblocks

Abstract

Gene expression in prokaryotes and eukaryotes is a process that requires a high level of regulation, with much of this regulation occurring at the level of transcription. The work described in this thesis focuses on a specific type of interaction that occurs during transcription elongation, where an RNA polymerase (RNAP) moving along a DNA template encounters static DNA-bound proteins, which may act as roadblocks to transcription. The two roadblock proteins studied in this thesis are the deactivated CRISPR associated Cas endonucleases dCas9 and dCas 12a. Both proteins are known to block transcription by RNAP. Systematic investigation of the Cas binding orientation bias and the influence of key parameters describing the RNAP- roadblock protein . interaction have advanced our understanding of general protein induced roadblocking and should assist in the development of specific and programmable roadblocking applications of Cas proteins (e.g., CRISPRi). Jn Chapters 3, 4 and 5, I successfully developed three important assays to investigate the behaviours of dCas9 and dCasl2a. Firstly, a roadblock assay was developed to measure roadblocking strength ;n vh10, where the dCas protein is targeted by a single guide RNA (sgRNA) to a transcribed region upstream of a Lacz reporter gene. The development of a curnate-inducible dCas expression system allowed for tuneabJe levels of roadblocking. Trials with guide sequence and target strand orientation confirmed differences in the orientation dependent nature of dCas9 and dCas l 2a roadblocking, and the importance of target sequence and position. Both dCas9 and dCas l 2a are better roadblocks when the RNAP first encounters the PAM end of the DNA-sgRNA-dCas complex. It was also demonstrated that changes in RNAP flux led to different roadblocking outcomes at equal roadblock protein concentrations, suggesting that transcribing RNAPs can dislodge the dCas proteins from the DNA. Secondly, a competition assay was developed to measure occupancy of roadblock protein binding sites in vivo, based on the ability of dCas to compete with the Lael repressor for binding at a Lac Oid operator and disrupt Lael mediated DNA looping. The results of this assay established the importance of the target sequence to the strength of dCas binding and how this then impacts on roadb locking activity. Fine tuning of the competition assay highlighted key features, such as the significant overlap between the dCas site and Oid operator required for a successful competition assay. Thirdly, a quantitative western blot was developed to accurately determine the cellular concentration of dCas9 and dCas12a and infer how binding kinetics impact roadblocking strength. In combination with the site occupancy data from the competition assay, knowledge of the cellular concentration allows calculation of the in vivo binding affinity of the dCas9 and dCasl2a proteins. Together, the data suggest that the dCas proteins have extremely high binding affinity in vivo, with dCas9 binding more tightly compared to dCasl2a. This higher affinity likely results from a combination of a moderately faster onrate constant and a moderately slower off-rate. An understanding of the relationships between dCas binding kinetics, site occupancy, RNAP flux and roadblock activity were developed. In Chapter 6, the effects of guide mismatches on dCasl2a competition and roadblocking were investigated. Guide mismatches introduced at the PAM proximal end tended to have a greater effect on dCasl2a binding than mismatches atthe distal end. Similarly, dCasl2a with guide mismatches at the PAM end were more readi ly dislodged by elongating RNAP, and thus had weakened roadblock activity. Overall, this work investigating dCas9 and dCas l2a-mediated roadblocking of transcription has allowed us to develop a better understanding of the mechanisms underlying proteinmediated roadblocking of transcription, paving the way for the utilisation of these interactions for a range of application.