Review of Homology-directed repair of a defective glabrous gene in Arabidopsis with Cas9-based gene targeting
Homology-directed repair of a defective glabrous gene in Arabidopsis with Cas9-based gene targeting [Florian Hahn, Marion Eisenhut, Otho Mantegazza, Andreas P.M. Weber, January 5, 2018, BioRxiv] [https://doi.org/10.1101/243675]Overview and take-home messages: Hahn et al. have compared the efficiencies of two different methods that have been previously reported to enhance the frequency of homologous recombination in plants. The paper has focused on testing a viral replicon system with two different enzymes, nuclease and nickase, as well as an in planta gene targeting (IPGT) system in Arabidopsis thaliana. Interestingly, authors have chosen GLABROUS1 (GL1), a regulator of trichome formation, as a visual marker to detect Cas9 activity and therefore homologous recombination. A 10 bp deletion in the coding region of GL1 gene produces plants devoid of trichomes. Out of the two methods in planta gene targeting approach successfully restored trichome formation in less than 0.2% of ~2,500 plants screened, whereas the method based on viral replicon machinery did not manage to restore trichome formation at all. This manuscript is of high quality, experiments are well designed and executed. However, there are some concerns that could be addressed in the next preprint or print version. Below are some feedback and suggestions that we hope will improve the manuscript.
PreReview Journal Club: Evolutionary dynamics of bacteria in the gut microbiome within and across hosts
Evolutionary dynamics of bacteria in the gut microbiome within and across hosts
Nandita R. Garud*, Benjamin H. Good*, Oskar Hallatschek, Katherine S.Pollard
Originally published on biorxiv
Comments on "An empirical test of the temperature dependence of carrying capacity"
This is a preprint review of An empirical test of the temperature dependence of carrying capacity by Joey Bernhardt, Jennifer M. Sunday, and Mary I. O'Connor. The preprint was originally posted on bioRxiv on October 28, 2017 (DOI: https://doi.org/10.1101/210690).
Preprints demystified: an interactive workshop at University College London
Hello! Thank you to all who came to our preprint workshop at UCL 30th October, 2017. We thoroughly enjoyed the discussion and we hope it was useful to you too. Due to the superb note-taking skills of Naomi Penfold, I am able to provide a detailed summary of the event as a refresher for those who came, and as info for those who couldn't make it. If you are interested in hosting your own preprint event and would like some help, please let me know (email@example.com). Enjoy!
Review of Medicago truncatula Zinc-Iron Permease6 provides zinc to rhizobia-infected nodule cells DOI: 10.1101/102426 (BioRxiv), 10.1111/pce.13035 (Plant Cell & Environment)
Medicago truncatula Zinc-Iron Permease6 provides zinc to rhizobia-infected nodule cells
[Isidro Abreu, Angela Saez, Rosario Castro-Rodriguez, Viviana Escudero, Benjamin Rodriguez-Haas, Marta Senovilla, Camille Laure, Daniel Grolimund, Manuel Tejada-Jimenez, Juan Imperial, Manuel Gonzalez-Guerrero , January 24, 2017 (preprint), September 21, 2017 (in print), BioRxiv & Wiley-Blackwell]
BIGC - PREreview of "Exotic species dominate due to niche overlap in a complex grassland community"
This is a preprint journal club review of "Exotic species dominate due to niche overlap in a complex grassland community" by Lawrence H. Uricchio, S. Caroline Daws, Erin R. Spear and Erin A. Mordecai. Te preprint was originally posted on bioRxiv on January 15, 2018 (DOI: https://doi.org/10.1101/253518) https://www.biorxiv.org/content/early/2018/01/24/253518. Our group Biotic Interactions and Global Change (BIGC) reviewed this paper in February 2018.
A CRISPR screening approach for identifying novel autophagy-related factors and cytoplasm-to-lysosome trafficking routes
This report was prepared by Vienna Biocenter Summer Class 2017 PhD students as a part of their Priming Your PhD training. Please see the instructors and participating students’ names at the end of the review. Shoemaker et al. performed a systematic genome wide CRISPR screen and identified a catalogue of factors involved in mammalian autophagy. They validated their findings by further characterizing one of the newly identified autophagy-related factors in their screen. They showed that TMEM41B, an integral ER-membrane protein is involved in maturation of the phagophore. Furthermore, using NBR1 as an autophagy substrate, they discovered a number of genes involved in a novel non-canonical autophagy pathway, which is independent of ATG7. The comprehensive results obtained in this study make the picture of mammalian autophagy more complete, and provide entry points for future studies that will dissect alternative autophagy pathways and the molecular mechanism underlying the role of TMEM41B in canonical autophagy. Genome wide CRISPR-screen provides a state of the art, unbiased approach to discover unknown genes in the autophagy process. They have developed a sensitive autophagic flux measurement reporter using the tandem-fluorescent reporters. In addition to the commonly used LC3B, they have also used known cargo receptors to identify autophagy regulators. Using this reporter system as a readout for a genome-wide screen is opening up the possibility to systematically dissect complex cellular autophagy networks. The authors were able to validate their screening approach by recovering almost all known ATG factors. Interestingly, the screen also identified a number of completely new candidates. While the authors have initially setup screens to address the pathways involved in the five major autophagy receptors, their last screening setup also allows to dissect the pathway involved in the highly debated ATG7-independent lysosomal targeting. We find this particularly interesting as is significantly contributes to our general understanding of how the network of autophagy receptors is organized. Furthermore, the description of ATG-independent alternative autophagy pathways questions the use of ATG7 KO genotypes as a control in autophagy experiments. As autophagy may still be active in ATG7 KO, this should be of concern for future experimental designs. The authors extensively stress-tested their reporter systems prior to performing the screen. They have also integrated all the reporters in the same loci to prevent expression level differences. The authors, in most cases, transparently provide numbers of cells used in the experimental setup and respective controls. Throughout the paper the authors confirm and cross-reference results obtained from other researchers in the field. They have used a broad range of experimental tools to validate their screen and results. Overall, the manuscript is of very high quality. Below are some suggestions that we hope will improve the manuscript. 1. The authors have chosen a 60% interval for their FACS sorting range. It would be useful if they provide the original FACS plots (RFP/GFP ratio), in order for us to see how the distribution of non-infected and library-infected populations look like. This would explain why screening for 60% of the population is necessary/reasonable. Although they have used a published sgRNA library, it would have been useful if they provided more information such as how many sgRNAs/gene are in the initial library, library representation in the transduced cell pool, replicate correlation plots. In addition, previous CRISPR screen papers have used fold changes for quantifying their hits. It would be useful if the authors explain the rationale behind beta scores and give more detail on beta score calculation. 2. There is also no information about Cas9 clonality in the K562 cells which were used for the screen. This would be useful for readers. 3. In the validation experiments, the authors state “mock treated cells” as negative control – does this mean that no sgRNA is transduced? If so, this is not an applicable negative control, as biological response to transduction and DNA damage repair by introducing an sgRNA is not addressed. Therefore, using a sgRNA targeting a gene desert would be the correct control. 4. Information how they normalized to 1.0 in Fig 3A is missing, as well as additional labels on Y-Axis that would enable the reader to understand whether this is a linear or log-scale. 5. In Figure 6, Stx17 and one of the cargo receptors could have been nice controls. Additionally, it would have been nice to have EM images of autophagosomes in TMEM41B KO cells. 6. Due to the design of the screen, one would have expected to get some hits related to lysosomal acidification. The authors did not mention this in the results or discussion. It would be nice to at least discuss this. Similarly, the authors should compare and contrast DeJesus et al., 2017 eLife paper in the discussion. This paper uses a similar CRISPR approach and looks for autophagy regulators. 7. In the introduction, it would have been beneficial to introduce the SQSTM1 receptors in a bit more detail, since the screen is based on them. 8. It would be useful if they clarified why they decided to use TMEM41B, since it wasn’t a very strong hit for all the receptors tested. 9. The description of the cell line used for the screen was unclear; were single clones or a bulk population used? 10. Figure S6 is missing; the supplementary figures go from Figure S5 to S7. 11. In the text, RAB7A and HOPS are mentioned while referring to figure 7E. However, these factors cannot be found in the figure. 12. The last paragraph of the results section refers to an analysis about genetic interactions between ATG7 and several factors, but no figure is ever referenced for this section. 13. Figure 3A: in the legend, more information could be given as to what exactly is plotted and less regarding the supplementary data. It is difficult to determine the scale of the Y-axis as only two values are given. PhD Students: Krista Briedis, Alexander Bykov, Claudia Ctortecka, Melanie De Almeida, Philipp Dexheimer, Joachim Garbrecht, Sarah Gruenbacher, Bence Hajdusits, Felix Holstein, Bhagyshree Jamge, Friederike Leesch, Joanna Nowacka, Mina Petrovic, Anna Schmuecker, Monika Steininger, Pietro Tardivo, Szu-Hsien Wu Facilitators: Fumiyo Ikeda, Yasin Dagdas
BIGC - PREreview of "Evolutionary responses to conditionality in species interactions across environmental gradients"
This is a preprint journal club review of "Evolutionary responses to conditionality in species interactions across environmental gradients" by Anna M O'Brien, Ruairidh J.H. Sawers, Jeffrey Ross-Ibarra, Sharon Y Strauss. The preprint was originally posted on bioRxiv on December 10, 2017 (DOI: https://doi.org/10.1101/031195) https://www.biorxiv.org/content/early/2017/12/10/031195.Our group Biotic Interactions and Global Change (BIGC) reviewed this paper on January, 2017 .
Review on INArxiv preprint: K-Means Method for Clustering Water Quality Status on The Rivers of Banjarmasin
This is a preprint journal club review of K-Means Method for Clustering Water Quality Status on The Rivers of Banjarmasin by Tien Zubaidah and Nieke Karnaningroem. The preprint was originally posted on INArxiv on December 21, 2017 (link: https://osf.io/g6wkp/). The article is now in review in the ARPN Journal of Engineering and Applied Sciences (submitted December 20, 2017). Original abstract: The surface river water quality in Banjarmasin city tends to decline constantly as the result of direct and indirect waste disposal from various human activities along the river body. This study aimed to determine the vulnerability points against pollution in the rivers of Banjarmasin using clustering techniques with K-means algorithm. The parameters observed include Biological Oxygen Demand (BOD), Chemical Oxygen Demand (COD), Total Suspend Solid (TSS) and Dissolved Oxygen (DO). The data were collected at eight water monitoring stations on various rivers in Banjarmasin city. With the K-means method, four water quality status were clustered. The result showed that 6 stations observed during the period April to October 2016 were categorized into the heavy polluted cluster with major pollution point of sources came from the domestic and industrial activities.