Cell-free biosensors for rapid detection of water contaminants.

Lack of access to safe drinking water is a global problem, and methods to reliably and easily detect contaminants could be transformative. We report the development of a cell-free in vitro transcription system that uses RNA Output Sensors Activated by Ligand Induction (ROSALIND) to detect contaminants in water. A combination of highly processive RNA polymerases, allosteric protein transcription factors and synthetic DNA transcription templates regulates the synthesis of a fluorescence-activating RNA aptamer. The presence of a target contaminant induces the transcription of the aptamer, and a fluorescent signal is produced. We apply ROSALIND to detect a range of water contaminants, including antibiotics, small molecules and metals. We also show that adding RNA circuitry can invert responses, reduce crosstalk and improve sensitivity without protein engineering. The ROSALIND system can be freeze-dried for easy storage and distribution, and we apply it in the field to test municipal water supplies, demonstrating its potential use for monitoring water quality.

Simultaneous repression of multiple bacterial genes using nonrepetitive extra-long sgRNA arrays.

Engineering cellular phenotypes often requires the regulation of many genes. When using CRISPR interference, coexpressing many single-guide RNAs (sgRNAs) triggers genetic instability and phenotype loss, due to the presence of repetitive DNA sequences. We stably coexpressed 22 sgRNAs within nonrepetitive extra-long sgRNA arrays (ELSAs) to simultaneously repress up to 13 genes by up to 3,500-fold. We applied biophysical modeling, biochemical characterization and machine learning to develop toolboxes of nonrepetitive genetic parts, including 28 sgRNA handles that bind Cas9. We designed ELSAs by combining nonrepetitive genetic parts according to algorithmic rules quantifying DNA synthesis complexity, sgRNA expression, sgRNA targeting and genetic stability. Using ELSAs, we created three highly selective phenotypes in Escherichia coli, including redirecting metabolism to increase succinic acid production by 150-fold, knocking down amino acid biosynthesis to create a multi-auxotrophic strain and repressing stress responses to reduce persister cell formation by 21-fold. ELSAs enable simultaneous and stable regulation of many genes for metabolic engineering and synthetic biology applications.