Multiplexed activation in mammalian cells using a split-intein CRISPR/Cas12a based synthetic transcription factor.

James W Bryson,Susan J Rosser,Jamie Y Auxillos

doi:10.1093/nar/gkab1191

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. The recently discovered CRISPR/Cas12a (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 the Francisella novicida Cas12a (FnCas12a) possessing a shorter PAM sequence than Acidaminococcus sp. (As) or Lachnospiraceae bacterium (Lb) variants, enabling denser targeting of genomic loci, while performing just as well or even better than the other variants. 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.

Highlights

Synthetic transcription factors are modular proteins composed of DNA binding domains and transactivation domains, which enable up-regulation of targeted genes
We have shown the first application of engineering FnCas12a derived synthetic transcription factors in mammalian cells
The key advantage of the Fn variant is the simpler protospacer adjacent motif (PAM) sequence ‘KYTV’ when compared to the commonly utilised Acidaminococcus sp. (As) or Lachnospiraceae bacterium (Lb) variants ‘TTTV’. This translates to being able to target on average every 21 nt as opposed to on average every 85 nt, highly comparable to the CRISPR associated protein 9 (Cas9) PAM sequence ‘NGG’ which enables targeting on average every 16 nt

Summary

Introduction

Synthetic transcription factors are modular proteins composed of DNA binding domains and transactivation domains, which enable up-regulation of targeted genes. A hybridised CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) enables targeting of the CRISPR associated protein 9 (Cas9) to a specific locus [3]. There must be a protospacer adjacent motif (PAM) sequence, which varies based on the Cas species of origin, adjacent to the target sequence. If the PAM sequence is present, Cas can transiently melt the DNA to enable infiltration by the spacer sequence [4]. If the spacer is complementary to the target sequence, Cas will bind and cleave the target DNA

Methods

Results

Conclusion