Bacterial autotrophs often rely on CO₂ concentrating mechanisms (CCMs) to assimilate carbon. Although many CCM proteins have been identified, a systematic screen of the components of CCMs is lacking. Here, we performed a genome-wide barcoded transposon screen to identify essential and CCM-related genes in the γ-proteobacterium Halothiobacillus neapolitanus. Screening revealed that the CCM comprises at least 17 and probably no more than 25 genes, most of which are encoded in 3 operons. Two of these operons (DAB1 and DAB2) contain a two-gene locus that encodes a domain of unknown function (Pfam: PF10070) and a putative cation transporter (Pfam: PF00361). Physiological and biochemical assays demonstrated that these proteins-which we name DabA and DabB, for DABs accumulate bicarbonate-assemble into a heterodimeric complex, which contains a putative β-carbonic anhydrase-like active site and functions as an energy-coupled inorganic carbon (C_i) pump. Interestingly, DAB operons are found in a diverse range of bacteria and archaea. We demonstrate that functional DABs are present in the human pathogens Bacillus anthracis and Vibrio cholerae. On the basis of these results, we propose that DABs constitute a class of energized C_i pumps and play a critical role in the metabolism of C_i throughout prokaryotic phyla.

Direct, amplification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental samples. CRISPR-Cas nucleases offer programmable RNA-guided RNA recognition that triggers cleavage and release of a fluorescent reporter molecule, but long reaction times hamper their detection sensitivity and speed. Here, we show that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ~30 molecules per µl of RNA in 20 min. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA extracted from respiratory swab samples with quantitative reverse transcriptase PCR (qRT-PCR)-derived cycle threshold (C_t) values up to 33, using a compact detector. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables sensitive, direct RNA detection in a format that is amenable to point-of-care infection diagnosis as well as to a wide range of other diagnostic or research applications.

Direct, amplification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental samples. CRISPR-Cas nucleases offer programmable RNA-guided recognition of RNA that triggers cleavage and release of a fluorescent reporter molecule 1,2 , but long reaction times hamper sensitivity and speed when applied to point-of-care testing. Here we show that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ∼30 RNA copies/microliter in 20 minutes. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that detected SARS-CoV-2 RNA from nasopharyngeal samples with PCR-derived Ct values up to 29 in microfluidic chips, using a compact imaging system. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables direct RNA detection in a format amenable to point-of-care infection diagnosis, as well as to a wide range of other diagnostic or research applications.

In the version of this Article initially published, there were errors in the author affiliations, Fig. 3, main text and Acknowledgements section.