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Microbial organisms are key drivers in processes related to human health, industrial biotechnology, and environmental ecology. In our attempts to predict, control, and design biological outcomes in each of these application areas, we require both an understanding of how microbes encode and perform their innate functions and the tools to implement engineering decisions. Here we present new technologies for interrogating genome function and editing genomes to carry out user-defined functions. First, we describe the development of CRISPRi as a platform for high-throughput genome annotation, focusing on a proof-of-concept in Escherichia coli to demonstrate our ability to map the genotype-phenotype-function landscape and uncover new design considerations for improving CRISPRi-based genetic screens. Next, we present the creation of a platform strain of Escherichia coli that achieves high rates of multiplex genome editing while retaining a low background mutagenesis rate, a favorable tradeoff that many editing strains have struggled to achieve. Finally, we report the design of novel CRISPR architectures that act not only as promising scaffolds for effector fusions but can also be programmed to sense and respond to endogenous cellular signals. Together, these technologies improve our ability to rapidly understand and engineer microbial behaviors.

Microbial organisms are key drivers in processes related to human health, industrial biotechnology, and environmental ecology. In our attempts to predict, control, and design biological outcomes in each of these application areas, we require both an understanding of how microbes encode and perform their innate functions and the tools to implement engineering decisions. Here we present new technologies for interrogating genome function and editing genomes to carry out user-defined functions. First, we describe the development of CRISPRi as a platform for high-throughput genome annotation, focusing on a proof-of-concept in Escherichia coli to demonstrate our ability to map the genotype-phenotype-function landscape and uncover new design considerations for improving CRISPRi-based genetic screens. Next, we present the creation of a platform strain of Escherichia coli that achieves high rates of multiplex genome editing while retaining a low background mutagenesis rate, a favorable tradeoff that many editing strains have struggled to achieve. Finally, we report the design of novel CRISPR architectures that act not only as promising scaffolds for effector fusions but can also be programmed to sense and respond to endogenous cellular signals. Together, these technologies improve our ability to rapidly understand and engineer microbial behaviors.