Robustness and innovation in synthetic genotype networks

Abstract

Genotype networks are sets of genotypes connected by small mutational changes that share the same phenotype. They facilitate evolutionary innovation by enabling the exploration of different neighborhoods in genotype space. Genotype networks, first suggested by theoretical models, have been empirically confirmed for proteins and RNAs. Comparative studies also support their existence for gene regulatory networks (GRNs), but direct experimental evidence is lacking. Here, we report the construction of three interconnected genotype networks of synthetic GRNs producing three distinct phenotypes in Escherichia coli. Our synthetic GRNs contain three nodes regulating each other by CRISPR interference and governing the expression of fluorescent reporters. The genotype networks, composed of over twenty different synthetic GRNs, provide robustness in face of mutations while enabling transitions to innovative phenotypes. Through realistic mathematical modeling, we quantify robustness and evolvability for the complete genotype-phenotype map and link these features mechanistically to GRN motifs. Our work thereby exemplifies how GRN evolution along genotype networks might be driving evolutionary innovation.

Design and Model-Driven Analysis of Synthetic Circuits with the Staphylococcus aureus Dead-Cas9 (sadCas9) as a Programmable Transcriptional Regulator in Bacteria

“…Regarding the steady-state output, a typical and well-known response is the bell-shaped curve as a function of the input concentration, used to generate patterns of gene expression, e.g., band-pass filters or French flag stripe patterning. , Another previously reported response of iFFL is a sigmoidal dose–response curve, when the circuit was designed for stabilizing gene expression, e.g., to counteract copy number changes, and it was important to obtain a constant steady-state output for wide ranges of input concentrations . Circuits implementing different types of iFFL motifs based on CRISPR/spdCas9 have been recently reported. , In this section, we designed an HSL-inducible iFFL circuitry in which sadCas9 and RFP were both placed downstream of P lux , and the sadOS site was assembled upstream of the RBS of RFP (Figure a). The circuit also had constitutive expression cassettes for LuxR, sgOS, and GFP.…”

“…Because of the throughput in construction and testing, a large number of circuit candidates can be produced, and, in the future, mathematical modeling may be rarely needed to support strain design, as suggested recently . However, modeling is still an essential step to reduce the design space when its exploration requires excessively large sequence combinations, and optimal solutions cannot be obtained from random libraries, or to support the design of circuits with improved robustness. , In both directions, the definition of new models like the ones illustrated in this work can support the investigation of unexpected behaviors of synthetic circuits. This knowledge will provide tools of increasing accuracy supporting the strain design process from the early design steps to debugging phases, highly relevant when automated DNA building/testing technologies are not available or when an in silico analysis of the feasible outputs of a network is needed to complement statistical or artificial intelligence methods in the interpretation of small or large data sets.…”

“…61 Circuits implementing different types of iFFL motifs based on CRISPR/spdCas9 have been recently reported. 14,62 In this section, we designed an HSL-inducible iFFL circuitry in which sadCas9 and RFP were both placed downstream of P lux , and the sadOS site was assembled upstream of the RBS of RFP (Figure 4a). The circuit also had constitutive expression cassettes for LuxR, sgOS, and GFP.…”

Synthetic Gene Circuits Combining CRISPR Interference and CRISPR Activation in E. coli: Importance of Equal Guide RNA Binding Affinities to Avoid Context-Dependent Effects

“…In this work, we successfully implemented the SoxS-based CRISPRa system first described by Fontana et al into our framework. , When we combined CRISPRa with CRISPRi, we observed a strong affinity competition for dCas9, leading to weak activation in on-target circuits and an incorrect circuit function of the off-target controls. This can lead to an undesirable coupling among circuit branches that theoretically should act orthogonal.…”

A direct experimental test of Ohno's hypothesis

“…This is challenging because of the recombinational instability inherent to repetitive DNA 6,43,44 . By drawing on our experience of using synthetic biology to study molecular evolution [45][46][47][48] , we designed a plasmid system that meets this challenge. It allowed us to evolve a fluorescent protein encoded by either double-copy or single-copy cogfp genes.…”