Multiplexed CRISPR technologies, in which numerous gRNAs or Cas enzymes are expressed at once, have facilitated powerful biological engineering applications, vastly enhancing the scope and efficiencies of genetic editing and transcriptional regulation. In this review, we discuss multiplexed CRISPR technologies and describe methods for the assembly, expression and processing of synthetic guide RNA arrays in vivo. Applications that benefit from multiplexed CRISPR technologies, including cellular recorders, genetic circuits, biosensors, combinatorial genetic perturbations, large-scale genome engineering and the rewiring of metabolic pathways, are highlighted. We also offer a glimpse of emerging challenges and emphasize experimental considerations for future studies.

Microbial consortia have been used in biotechnology processes, including fermentation, waste treatment, and agriculture, for millennia. Today, synthetic biologists are increasingly engineering microbial consortia for diverse applications, including the bioproduction of medicines, biofuels, and biomaterials from inexpensive carbon sources. An improved understanding of natural microbial ecosystems, and the development of new tools to construct synthetic consortia and program their behaviors, will vastly expand the functions that can be performed by communities of interacting microorganisms. Here, we review recent advancements in synthetic biology tools and approaches to engineer synthetic microbial consortia, discuss ongoing and emerging efforts to apply consortia for various biotechnological applications, and suggest future applications. Microbial Communities: An Emerging Paradigm in Synthetic Biology Microbial consortia (see Glossary) or communities are ubiquitous in nature and useful in many areas of the bioeconomy [1]. Natural consortia are important in the production of foods, the recycling of micronutrients, and in maintaining the health of humans, animals, and plants [2]. Such microbial communities consist of member organisms that, together, are more robust to environmental challenges, exhibit reduced metabolic burden due to a division of labor (DOL) and exchange of resources, possess expanded metabolic capabilities relative to monocultures, and can communicate (chemically or physically) between species [3-5]. Highlights Microbial consortia exhibit advantages over monocultures, including division of labor, spatial organization, and robustness to perturbations.

11 12 CRISPR is a versatile technology for genomic editing and regulation, but the 13 expression of multiple gRNAs in S. cerevisiae has thus far been limited. We present 14 here a simple extension to the Yeast MoClo Toolkit, which enables the rapid assembly 15 of gRNA arrays using a minimal set of parts. Using a dual-PCR, Type IIs restriction 16 enzyme Golden Gate assembly approach, at least 12 gRNAs can be assembled and 17 expressed from a single transcriptional unit. We demonstrate that these gRNA arrays 18 can stably regulate gene expression in a synergistic manner via dCas9-mediated 19 repression. This approach expands the number of gRNAs that can be expressed in 20 this model organism and may enable the versatile editing or transcriptional regulation 21 of a greater number of genes in vivo.

Advances in DNA sequencing have revolutionized our ability to read genomes. However, even in the most well-studied of organisms, the bacterium Escherichia coli, for ≈ 65% of promoters we remain ignorant of their regulation. Until we crack this regulatory Rosetta Stone, efforts to read and write genomes will remain haphazard. We introduce a new method, Reg-Seq, that links massively-parallel reporter assays with mass spectrometry to produce a base pair resolution dissection of more than 100 E. coli promoters in 12 growth conditions. We demonstrate that the method recapitulates known regulatory information. Then, we examine regulatory architectures for more than 80 promoters which previously had no known regulatory information. In many cases, we also identify which transcription factors mediate their regulation. This method clears a path for highly multiplexed investigations of the regulatory genome of model organisms, with the potential of moving to an array of microbes of ecological and medical relevance.

CRISPR gene activation and inhibition (CRISPRai) has become a powerful synthetic tool for influencing the expression of native genes for foundational studies, cellular reprograming, and metabolic engineering. Here we develop a method for near leak-free, inducible expression of a polycistronic array containing up to 24 gRNAs from two orthogonal CRISPR/Cas systems to increase CRISPRai multiplexing capacity and target gene flexibility. To achieve strong inducibility, we create a technology to silence gRNA expression within the array in the absence of the inducer, since we found that long gRNA arrays for CRISPRai can express themselves even without promoter. Using this method, we create a highly tuned and easy-to-use CRISPRai toolkit in the industrially relevant yeast, Saccharomyces cerevisiae, establishing the first system to combine simultaneous activation and repression, large multiplexing capacity, and inducibility. We demonstrate this toolkit by targeting 11 genes in central metabolism in a single transformation, achieving a 45-fold increase in succinic acid, which could be precisely controlled in an inducible manner. Our method offers a highly effective way to regulate genes and rewire metabolism in yeast, with principles of gRNA array construction and inducibility that should extend to other chassis organisms.

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Insulin and insulin-like growth factor 1 (IGF1) signaling is transduced by insulin receptor substrate 1 (IRS1) and IRS2. To elucidate physiological and redundant roles of insulin and IGF1 signaling in adult hearts, we generated mice with inducible cardiomyocyte-specific deletion of insulin and IGF1 receptors or IRS1 and IRS2.

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