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1. Escherichia coli cells are primed for survival before lethal antibiotic stress

   https://doi.org/10.1128/spectrum.01219-23  

   Hossain, Tahmina ; Singh, Abhyudai ; Butzin, Nicholas C. ( October 2023 , Microbiology Spectrum)

   Pagliara, Stefano (Ed.)

   Non-genetic factors can cause significant fluctuations in gene expression levels. Regardless of growing in a stable environment, this fluctuation leads to cell-to-cell variability in an isogenic population. This phenotypic heterogeneity allows a tiny subset of bacterial cells in a population called persister cells to tolerate long-term lethal antibiotic effects by entering into a non-dividing, metabolically repressed state. We occasionally noticed a high variation in persister levels, and to explore this, we tested clonal populations starting from a single cell using a modified Luria-Delbrück fluctuation test. Although we kept the conditions same, the diversity in persistence level among clones was relatively consistent: varying from ~60- to 100- and ~40- to 70-fold for ampicillin and apramycin, respectively. Then, we divided and diluted each clone to observe whether the same clone had comparable persister levels for more than one generation. Replicates had similar persister levels even when clones were divided, diluted by 1:20, and allowed to grow for approximately five generations. This result explicitly shows a cellular memory passed on for generations and eventually lost when cells are diluted to 1:100 and regrown (>seven generations). Our result demonstrates (1) the existence of a small population prepared for stress (“primed cells”) resulting in higher persister numbers; (2) the primed memory state is reproducible and transient, passed down for generations but eventually lost; and (3) a heterogeneous persister population is a result of a transiently primed reversible cell state and not due to a pre-existing genetic mutation.

   Antibiotics have been highly effective in treating lethal infectious diseases for almost a century. However, the increasing threat of antibiotic resistance is again causing these diseases to become life-threatening. The longer a bacteria can survive antibiotics, the more likely it is to develop resistance. Complicating matters is that non-genetic factors can allow bacterial cells with identical DNA to gain transient resistance (also known as persistence). Here, we show that a small fraction of the bacterial population called primed cells can pass down non-genetic information (“memory”) to their offspring, enabling them to survive lethal antibiotics for a long time. However, this memory is eventually lost. These results demonstrate how bacteria can leverage differences among genetically identical cells formed through non-genetic factors to form primed cells with a selective advantage to survive antibiotics.

    

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2. Transcriptional programming in a Bacteroides consortium

   https://doi.org/10.1038/s41467-022-31614-8  

   Huang, Brian D. ; Groseclose, Thomas M. ; Wilson, Corey J. ( July 2022 , Nature Communications)

   Bacteroidesspecies are prominent members of the human gut microbiota. The prevalence and stability ofBacteroidesin humans make them ideal candidates to engineer as programmable living therapeutics. Here we report a biotic decision-making technology in a community ofBacteroides(consortium transcriptional programming) with genetic circuit compression. Circuit compression requires systematic pairing of engineered transcription factors with cognate regulatable promoters. In turn, we demonstrate the compression workflow by designing, building, and testing all fundamental two-input logic gates dependent on the inputs isopropyl-β-D-1-thiogalactopyranoside and D-ribose. We then deploy complete sets of logical operations in five human donorBacteroides, with which we demonstrate sequential gain-of-function control in co-culture. Finally, we couple transcriptional programs with CRISPR interference to achieve loss-of-function regulation of endogenous genes—demonstrating complex control over community composition in co-culture. This work provides a powerful toolkit to program gene expression inBacteroidesfor the development of bespoke therapeutic bacteria.

    

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3. Independent control of mean and noise by convolution of gene expression distributions

   https://doi.org/10.1038/s41467-021-27070-5  

   Gerhardt, Karl P. ; Rao, Satyajit D. ; Olson, Evan J. ; Igoshin, Oleg A. ; Tabor, Jeffrey J. ( November 2021 , Nature Communications)

   Gene expression noise can reduce cellular fitness or facilitate processes such as alternative metabolism, antibiotic resistance, and differentiation. Unfortunately, efforts to study the impacts of noise have been hampered by a scaling relationship between noise and expression level from individual promoters. Here, we use theory to demonstrate that mean and noise can be controlled independently by expressing two copies of a gene from separate inducible promoters in the same cell. We engineer low and high noise inducible promoters to validate this result inEscherichia coli, and develop a model that predicts the experimental distributions. Finally, we use our method to reveal that the response of a promoter to a repressor is less sensitive with higher repressor noise and explain this result using a law from probability theory. Our approach can be applied to investigate the effects of noise on diverse biological pathways or program cellular heterogeneity for synthetic biology applications.

    

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4. Identifying and retargeting transcriptional hot spots in the human genome

   https://doi.org/10.1002/biot.201600015  

   Cheng, Joseph K. ; Lewis, Amanda M. ; Kim, Do Soon ; Dyess, Timothy ; Alper, Hal S. ( June 2016 , Biotechnology Journal)

   Mammalian cell line development requires streamlined methodologies that will reduce both the cost and time to identify candidate cell lines. Improvements in site‐specific genomic editing techniques can result in flexible, predictable, and robust cell line engineering. However, an outstanding question in the field is the specific site of integration. Here, we seek to identify productive loci within the human genome that will result in stable, high expression of heterologous DNA. Using an unbiased, random integration approach and a green fluorescent reporter construct, we identify ten single‐integrant, recombinant human cell lines that exhibit stable, high‐level expression. From these cell lines, eight unique corresponding integration loci were identified. These loci are concentrated in non‐protein coding regions or intronic regions of protein coding genes. Expression mapping of the surrounding genes reveals minimal disruption of endogenous gene expression. Finally, we demonstrate that targeted de novo integration at one of the identified loci, the 12^thexon‐intron region of theGRIK1gene on chromosome 21, results in superior expression and stability compared to the standard, illegitimate integration approach at levels approaching 4‐fold. The information identified here along with recent advances in site‐specific genomic editing techniques can lead to expedited cell line development.

    

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5. High‐efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR /Cas9

   https://doi.org/10.1111/tpj.13446  

   Gil‐Humanes, Javier ; Wang, Yanpeng ; Liang, Zhen ; Shan, Qiwei ; Ozuna, Carmen V. ; Sánchez‐León, Susana ; Baltes, Nicholas J. ; Starker, Colby ; Barro, Francisco ; Gao, Caixia ; et al ( February 2017 , The Plant Journal)

   The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence‐specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved usingAgrobacteriumT‐DNA, biolistics or by stably integrating nuclease‐encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such asNicotinana tabacum(tobacco) andSolanum lycopersicum(tomato), greater than 10‐fold enhancements inGTfrequencies have been achieved usingDNAvirus‐based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundantSSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon‐based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110‐fold increase in expression of a reporter gene relative to non‐replicating controls. Furthermore, replicons carryingCRISPR/Cas9 nucleases and repair templates achievedGTat an endogenousubiquitinlocus at frequencies 12‐fold greater than non‐viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these highGTfrequencies. We also demonstrate gene‐targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with theWDVreplicons, multiplexedGTwithin the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies ofGTusingWDV‐basedDNAreplicons will make it possible to edit complex cereal genomes without the need to integrateGTreagents into the genome.

    

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