Orthogonal Modular Gene Repression in Escherichia coli Using Engineered CRISPR/Cas9

Abstract

The progress in development of synthetic gene circuits has been hindered by the limited repertoire of available transcription factors. Recently, it has been greatly expanded using the CRISPR/Cas9 system. However, this system is limited by its imperfect DNA sequence specificity, leading to potential crosstalk with host genome or circuit components. Furthermore, CRISPR/Cas9-mediated gene regulation is context dependent, affecting the modularity of Cas9 based transcription factors. In this paper we address the problems of specificity and modularity by developing a computational approach for selecting Cas9/gRNA transcription factor/promoter pairs that are maximally orthogonal to each other as well as to the host genome and synthetic circuit components. We validate the method by designing and experimentally testing four orthogonal promoter/repressor pairs in the context of a strong promoter PL from phage lambda. We demonstrate that these promoters can be interfaced by constructing a double and a triple inverter circuits. To address the problem of modularity we propose and experimentally validate a scheme to predictably incorporate orthogonal CRISPR/Cas9 regulation into a large class of natural promoters.

CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942

“…CRISPRi holds great promise for a wide range of applications in microorganisms, including bacterial cell growth control [35], genetic screen [25, 36], synthetic biology module development [37, 38] or metabolic networks control in various microorganisms such as E. coli [24, 39, 40], mycobacteria [41], Bacillus subtilis [42], Corynebacterium glutamicum [43], Clostridium beijerinckii [44], yeast [45] and cyanobacteria [7]. In particular, a number of recent studies have exploited CRISPRi to regulate the metabolic pathways in E. coli for enhanced production of various biotechnological products including poly(3-hydroxybutyrate- co -4-hydroxybutyrate) [23], terpenoid [8], pinosylvin [46], flavonoid [47] and mevalonate [48].…”

Transcriptional regulation with CRISPR-Cas9: principles, advances, and applications

“…The repression strength is strongly dependent on the position with respect to the target promoter as well as the nature of promoter itself [7,8,10,11]. In prokaryotes , repression of up to 1000-fold was achieved when targeting dCas9 to either DNA strand within a promoter or to the non-template DNA strand downstream [7,8,10,15–18].…”

“…Chromatin state also affects dCas9 DNA binding: it is much weaker within condensed chromatin regions, therefore very good match between sgRNA and target DNA sequence is required [9,46,52]. Further studies of dCas9 and specificity-improved Cas9 proteins [53,54] in the aforementioned contexts will inform further development of computational algorithms [11,43,51,52,55] that incorporate these effects to precisely select the types and amounts of sgRNAs that are most appropriate to specific applications.…”

“…First, naïve choice of sgRNA guide sequences can result in undesired crosstalk that leads to interference with the circuit or host function [8]. This problem can be addressed by computationally designing sgRNA guide sequences and the corresponding target DNA sites in order to minimize undesired interactions [11]. Second, while dCas9/sgRNA complex binds its cognate target DNA very tightly, the dissociation kinetics is slow (half-life of ~6hrs or more in vitro [45,60]).…”

“…(a) Orthogonal modular dCas9-repressible promoters have been designed for building synthetic gene circuits in Escherichia coli . (b) Proof of principle logic circuits were built using the promoters shown in (a) [10,11]. (c) dCas9-repressible (top) and dCas9-activatable (bottom) eukaryotic modular promoters have been designed for building synthetic circuits in eukaryotes.…”