Abstract
The adoption of CRISPR systems for the generation of synthetic transcription factors has greatly simplified the process for upregulating endogenous gene expression, with a plethora of applications in cell biology, bioproduction and cell reprogramming. In particular the recently discovered Cas12a systems offer extended potential, as Cas12a is capable of processing its own crRNA array to provide multiple individual crRNAs for subsequent targeting from a single transcript. Here we show the application of dFnCas12a-VPR in mammalian cells, with FnCas12a possessing a shorter PAM sequence than As or Lb variants, enabling denser targeting of genomic loci. We observe that synergistic activation and multiplexing can be achieved using crRNA arrays but also show that crRNAs expressed towards the 5’ of 6-crRNA arrays show evidence of enhanced activity. This not only represents a more flexible tool for transcriptional modulation but further expands our understanding of the design capabilities and limitations when considering longer crRNA arrays for multiplexed targeting.
Competing Interest Statement
The authors have declared no competing interest.
