Design and Implementation of Reprogrammable dCas9 Transcription

Factor Wires

Miles W. Gander*, William Voje Jr.â? , James Carothersâ? , Eric Klavins*

*Department of Electrical Engineering, University of Washington.

â? Department of Chemical Engineering, University of Washington.

Keywords: CRISPR, Synthetic Gene Regulation, RNA-guided multiplex gene regulation, RNA

folding design, Ribozymes

Presently, the size and complexity of engineered synthetic gene networks is limited by the small number well-characterized and robust genetic components. Here, we introduce and demonstrate a framework for creating orthogonal repressing transcription factor wires which exploit the programmability of the CRISPR/Cas9 system in S. cerevisiae. Genetic logic circuits such as NOR gates and layered cascades have been implemented in bacteria and eukaryotes using CRISPRi, but are limited by non-cooperative response functions of the guide RNA (gRNA), leading to leak and signal degradation as the
number of layers increase [1,2]. To achieve leak free off states, we use a eukaryotic system and a dCas9 fused with a strong chromatin remodeling repression domain (RD). In our system, a transcription factor wire consists of 1) a dCas9-RD fusion protein that when recruited to DNA represses transcription of its target and 2) a guide RNA (gRNA) which when complexed with the dCas9-RD binds a specific DNA sequence. These wires link the output of Pol II gRNA expression cassettes to their cognate gRNA responsive
Pol II promoters (pGRRs). Pol II promoters are used as wire nodes, instead of non- coding RNA Pol III promoters, because of their variety and engineerability in S. cerevisiae [3]. To consistently express gRNA from Pol II promoters novel computational design methodologies, which consider minimum free energy and kinetic simulations of RNA structure, are used to augment and extend existing ribozyme-flanked gRNA expression methods [4]. Our synthetic pGRRs contain two discrete gRNA target sites and function as NOR gates. Therefore, systems of wires with minimal crosstalk can create any logic function. Building an orthogonal set of gRNAs and pGRRs is challenging due to the promiscuity of dCas9-gRNA complexes formed with imperfect DNA complements [5,6]. We developed a computational design strategy that generates sets of functional and orthogonal transcription factor wire libraries, enabling the construction of larger and more complex gene networks. To demonstrate the functionality of these wires, we created a library of 20 NOR gates and created a 4- component sequential repression cascade in S. cerevisiae. This work advances the
state of the art for cellular logic and provides a framework for creating more complex circuits.

References

[1] Nielsen, Alec AK, and Christopher A. Voigt. "Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks." Molecular systems biology10.11 (2014).

[2] Kiani, Samira, et al. "CRISPR transcriptional repression devices and layered circuits in mammalian cells." Nature methods (2014).

[3] Blazeck, John, et al. "Controlling promoter strength and regulation in Saccharomyces cerevisiae using synthetic hybrid promoters." Biotechnology and bioengineering 109.11 (2012): 2884-2895.

[4] Gao, Yangbin and Yunde Zhao. â??Self-Processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing.â? Journal of integrative plant biology 56.4 (2014): 343-349.

[5] Mali, Prashant, et al. "CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering." Nature biotechnology (2013).

[6] Doench, John G., et al. "Rational design of highly active sgRNAs for CRISPR-Cas9- mediated gene inactivation." Nature biotechnology (2014)