Topics overview: What’s Metafluidics; How to solve the Cpf1 function limitation in BV3L6 and Lachnospiraceae bacterium ND2006; The breakthrough of single-cell genome sequencing to large cell populations; Bacteria can be engineered to function as diagnostics or therapeutics in the mammalian gut; TACCA was applied to produce a high-quality (N50 of 9.76 Mb) de novo chromosome assembly of the wheat line CH Campala Lr22a in only 4 months.
1. Open-source, community-driven microfluidics with Metafluidics.
Microfluidic devices have the potential to automate and miniaturize biological experiments, but open-source sharing of device designs has lagged behind sharing of other resources such as software. Synthetic biologists have used microfluidics for DNA assembly, cell-free expression, and cell culture, but a combination of expense, device complexity, and reliance on custom set-ups hampers their widespread adoption. David S Kong at Massachusetts Institute of Technology Lincoln Laboratory in Massachusetts, USA and his colleagues present Metafluidics, an open-source, community-driven repository that hosts digital design files, assembly specifications, and open-source software to enable users to build, configure, and operate a microfluidic device. They use Metafluidics to share designs and fabrication instructions for both a microfluidic ring-mixer device and a 32-channel tabletop microfluidic controller. This device and controller are applied to build genetic circuits using standard DNA assembly methods including ligation, Gateway, Gibson, and Golden Gate. Metafluidics is intended to enable a broad community of engineers, DIY enthusiasts, and other nontraditional participants with limited fabrication skills to contribute to microfluidic research.
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2. Engineered Cpf1 variants with altered PAM specificities.
The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, Linyi Gao at Broad Institute of MIT and Harvard in Massachusetts, USA and his colleagues performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. They engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS indicated that these variants retain high DNA-targeting specificity, which they further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified PAM-interacting mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately threefold in human coding sequences to one cleavage site per ~11 bp.
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3. Single-cell genome sequencing at ultra-high-throughput with microfluidic droplet barcoding.
The application of single-cell genome sequencing to large cell populations has been hindered by technical challenges in isolating single cells during genome preparation. Here Freeman Lan at University of California in California, USA and his colleagues present single-cell genomic sequencing (SiC-seq), which uses droplet microfluidics to isolate, fragment, and barcode the genomes of single cells, followed by Illumina sequencing of pooled DNA. They demonstrate ultra-high-throughput sequencing of >50,000 cells per run in a synthetic community of Gram-negative and Gram-positive bacteria and fungi. The sequenced genomes can be sorted in silico based on characteristic sequences. They use this approach to analyze the distributions of antibiotic-resistance genes, virulence factors, and phage sequences in microbial communities from an environmental sample. The ability to routinely sequence large populations of single cells will enable the de-convolution of genetic heterogeneity in diverse cell populations.
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4. Engineered bacteria can function in the mammalian gut long-term as live diagnostics of inflammation.
Bacteria can be engineered to function as diagnostics or therapeutics in the mammalian gut but commercial translation of technologies to accomplish this has been hindered by the susceptibility of synthetic genetic circuits to mutation and unpredictable function during extended gut colonization. Here, David T Riglar at Harvard Medical School in Massachusetts, USA and his colleagues report stable, engineered bacterial strains that maintain their function for 6 months in the mouse gut. They engineered a commensal murine Escherichia coli strain to detect tetrathionate, which is produced during inflammation. Using their engineered diagnostic strain, which retains memory of exposure in the gut for analysis by fecal testing, they detected tetrathionate in both infection-induced and genetic mouse models of inflammation over 6 months. The synthetic genetic circuits in the engineered strain were genetically stable and functioned as intended over time. The durable performance of these strains confirms the potential of engineered bacteria as living diagnostics.
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5. Rapid cloning of genes in hexaploid wheat using cultivar-specific long-range chromosome assembly.
Cereal crops such as wheat and maize have large repeat-rich genomes that make cloning of individual genes challenging. Moreover, gene order and gene sequences often differ substantially between cultivars of the same crop species. A major bottleneck for gene cloning in cereals is the generation of high-quality sequence information from a cultivar of interest. In order to accelerate gene cloning from any cropping line, Anupriya Kaur Thind at University of Zurich in Zurich, Switzerl and and his colleagues report ‘targeted chromosome-based cloning via long-range assembly’ (TACCA). TACCA combines lossless genome-complexity reduction via chromosome flow sorting with Chicago long-range linkage to assemble complex genomes. They applied TACCA to produce a high-quality (N50 of 9.76 Mb) de novo chromosome assembly of the wheat line CH Campala Lr22a in only 4 months. Using this assembly they cloned the broad-spectrum Lr22a leaf-rust resistance gene, using molecular marker information and ethyl methanesulfonate (EMS) mutants, and found that Lr22a encodes an intracellular immune receptor homologous to the Arabidopsis thaliana RPM1 protein.
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