Generating and characterising Cas12a derived synthetic transcription factors
Bryson, James William
Transcription factors represent one of the primary determinants of gene expression changes and phenotypic modulation during dynamic processes in multi-cellular organisms. This includes processes as diverse and important as; development, differentiation and oncogenesis. Traditional scientific approaches rely on introducing one modification/change at a time and seeing how this impacts a specific outcome. As such, when probing the role from over-expression of a target gene, information can be acquired by over-expressing cDNA derived from a protein encoding gene. However such approaches remove much of the vital biological context provided by the local epigenetic context as well as the role of un-translated regions in modulating the stability, expression and localisation of transcripts/proteins. Conversely when over-expressing a transcription factor known to target the promoter of a gene of interest, it can become extremely challenging to narrow down the contribution to phenotype caused by the gene of interest being over-expressed, compared to the multiple other gene promoters bound by any one transcription factor. As such the generation of synthetic transcription factors providing predictable, stable binding to a specific locus, with minimal off-target binding provides powerful capabilities for exploration and manipulation of transcriptional networks. To this end, RNA guided homing endonuclease CRISPR systems have been adapted, through the generation of catalytically inactive variants, to serve as easily targetable DNA binding domains. These can be used to recruit transactivation domains to targeted promoters and increase expression for the target gene. In this work we validate and characterise synthetic transcription factors adapted from Cas12a/Cpf1 derived from three different species. Cas12a, unlike Cas9 possesses the ability to process its crRNA array through native RNase activity and uses the short crRNAs generated for the targeting of multiple unique loci. This means that designing and constructing genetic constructs for generating guide RNAs becomes cheaper and simpler and furthermore, the reduced size of DNA required for targeting provides benefits when considering packaging size constraints, such as with the AAV virus. We go on to further characterise the capabilities and limitations of these crRNA arrays for a variant derived from Francisella novicida - FnCas12a. This variant, unlike the more commonly utilised variants derived from Acidaminococcus sp BV3L6 (AsCas12a) and Lachnospiraceae bacterium ND2006 (LbCas12a), requires a shorter characterised PAM sequence ‘TTV’ for targeting, providing a comparable targeting density to the widely used Streptococcus pyogenes derived SpCas9 platforms. Our results open up dFnCas12a as a potential new gold standard for homing endonuclease derived scaffolds for recruiting diverse effector domains, preserving the best qualities of the SpCas9 system, whilst incorporating added strengths alongside the innate ability to operate orthogonally to existing SpCas9 systems.