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      Centriole and Golgi microtubule nucleation are dispensable for the migration of human neutrophil-like cells

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          Abstract

          Neutrophils migrate in response to chemoattractants to mediate host defense. Chemoattractants drive rapid intracellular cytoskeletal rearrangements including the radiation of microtubules from the microtubule-organizing center (MTOC) toward the rear of polarized neutrophils. Microtubules regulate neutrophil polarity and motility, but little is known about the specific role of MTOCs. To characterize the role of MTOCs on neutrophil motility, we depleted centrioles in a well-established neutrophil-like cell line. Surprisingly, both chemical and genetic centriole depletion increased neutrophil speed and chemotactic motility, suggesting an inhibitory role for centrioles during directed migration. We also found that depletion of both centrioles and GM130-mediated Golgi microtubule nucleation did not impair neutrophil directed migration. Taken together, our findings demonstrate an inhibitory role for centrioles and a resilient MTOC system in motile human neutrophil-like cells.

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          Most cited references42

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          Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR

          Background The success of the CRISPR/Cas9 genome editing technique depends on the choice of the guide RNA sequence, which is facilitated by various websites. Despite the importance and popularity of these algorithms, it is unclear to which extent their predictions are in agreement with actual measurements. Results We conduct the first independent evaluation of CRISPR/Cas9 predictions. To this end, we collect data from eight SpCas9 off-target studies and compare them with the sites predicted by popular algorithms. We identify problems in one implementation but found that sequence-based off-target predictions are very reliable, identifying most off-targets with mutation rates superior to 0.1 %, while the number of false positives can be largely reduced with a cutoff on the off-target score. We also evaluate on-target efficiency prediction algorithms against available datasets. The correlation between the predictions and the guide activity varied considerably, especially for zebrafish. Together with novel data from our labs, we find that the optimal on-target efficiency prediction model strongly depends on whether the guide RNA is expressed from a U6 promoter or transcribed in vitro. We further demonstrate that the best predictions can significantly reduce the time spent on guide screening. Conclusions To make these guidelines easily accessible to anyone planning a CRISPR genome editing experiment, we built a new website (http://crispor.org) that predicts off-targets and helps select and clone efficient guide sequences for more than 120 genomes using different Cas9 proteins and the eight efficiency scoring systems evaluated here. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-1012-2) contains supplementary material, which is available to authorized users.
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            High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities.

            The ability to perturb genes in human cells is crucial for elucidating gene function and holds great potential for finding therapeutic targets for diseases such as cancer. To extend the catalog of human core and context-dependent fitness genes, we have developed a high-complexity second-generation genome-scale CRISPR-Cas9 gRNA library and applied it to fitness screens in five human cell lines. Using an improved Bayesian analytical approach, we consistently discover 5-fold more fitness genes than were previously observed. We present a list of 1,580 human core fitness genes and describe their general properties. Moreover, we demonstrate that context-dependent fitness genes accurately recapitulate pathway-specific genetic vulnerabilities induced by known oncogenes and reveal cell-type-specific dependencies for specific receptor tyrosine kinases, even in oncogenic KRAS backgrounds. Thus, rigorous identification of human cell line fitness genes using a high-complexity CRISPR-Cas9 library affords a high-resolution view of the genetic vulnerabilities of a cell.
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              Neutrophil migration in infection and wound repair: going forward in reverse.

              Neutrophil migration and its role during inflammation has been the focus of increased interest in the past decade. Advances in live imaging and the use of new model systems have helped to uncover the behaviour of neutrophils in injured and infected tissues. Although neutrophils were considered to be short-lived effector cells that undergo apoptosis in damaged tissues, recent evidence suggests that neutrophil behaviour is more complex and, in some settings, neutrophils might leave sites of tissue injury and migrate back into the vasculature. The role of reverse migration and its contribution to resolution of inflammation remains unclear. In this Review, we discuss the different cues within tissues that mediate neutrophil forward and reverse migration in response to injury or infection and the implications of these mechanisms to human disease.
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                Author and article information

                Contributors
                Role: Monitoring Editor
                Journal
                Mol Biol Cell
                Mol Biol Cell
                molbiolcell
                mbc
                mboc
                Molecular Biology of the Cell
                The American Society for Cell Biology
                1059-1524
                1939-4586
                15 August 2021
                : 32
                : 17
                : 1545-1556
                Affiliations
                [a ]Molecular and Cellular Pharmacology Graduate Training Program, University of Wisconsin-Madison, Madison, WI 53706
                [b ]Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI 53706
                [c ]Medical Scientist Training Program, University of Wisconsin-Madison, Madison, WI 53706
                [d ]Department of Medicine, Division of Hematology/Oncology, University of Wisconsin-Madison, Madison, WI 53706
                [e ]Department of Pediatrics, University of Wisconsin-Madison, Madison, WI 53706
                University of California, Santa Barbara
                Author notes

                Present address: Department of Chemical and Biological Engineering, University of Colorado–Boulder, Boulder, CO 80303.

                *Address corrospondence to: Anna Huttenlocher ( huttenlocher@ 123456wisc.edu ).
                Article
                E21-02-0060
                10.1091/mbc.E21-02-0060
                8351748
                34191538
                d51b38ad-a028-4a4c-8605-34864967c17c
                © 2021 Klemm et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

                This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License.

                History
                : 05 February 2021
                : 27 May 2021
                : 11 June 2021
                Categories
                Brief Reports

                Molecular biology
                Molecular biology

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