Biology subjects

Kim, J.-S.

Publications and source records attributed to Kim, J.-S..

10 recordsLinked to original sources

bioRxiv2018

Phage Mediate Bacterial Self Recognition

Cells are social, and self-recognition is an important and conserved aspect of group behavior where cells assist kin and antagonize non-kin to conduct group behavior such as foraging for food and biofilm formation. However, the role of the common bacterial cohabitant, phage, in kin recognition, has not been explored. Here we find that a boundary (demarcation line) is formed between different swimming Escherichia coli strains but not between identical clones; hence, motile bacterial cells discriminate between self and non-self. The basis for this self-recognition is a novel, 49 kb, T1-type, lytic phage of the family siphoviridae (named here SW1) that controls formation of the demarcation line by utilizing one of the hosts cryptic prophage proteins, YfdM, to propagate. Critically, SW1 increases the fitness of E. coli K-12 compared to the identical strain that lacks the phage. Therefore, bacteria use phage to recognize kin.

molecular biology↗

bioRxiv2018

Web-based design and analysis tools for CRISPR base editing

BackgroundAs a result of its simplicity and high efficiency, the CRISPR-Cas system has been widely used as a genome editing tool. Recently, CRISPR base editors, which consist of deactivated Cas9 (dCas9) or Cas9 nickase (nCas9) linked with a cytidine or a guanine deaminase, have been developed. Base editing tools will be very useful for gene correction because they can produce highly specific DNA substitutions without the introduction of any donor DNA, but dedicated web-based tools to facilitate the use of such tools have not yet been developed.\n\nResultsWe present two web tools for base editors, named BE-Designer and BE-Analyzer. BE-Designer provides all possible base editor target sequences in a given input DNA sequence with useful information including potential off-target sites. BE-Analyzer, a tool for assessing base editing outcomes from next generation sequencing (NGS) data, provides information about mutations in a table and interactive graphs. Furthermore, because the tool runs client-side, large amounts of targeted deep sequencing data (>100MB) do not need to be uploaded to a server, substantially reducing running time and increasing data security. BE-Designer and BE-Analyzer can be freely accessed at http://www.rgenome.net/bedesigner/ and http://www.rgenome.net/be-analyzer/respectively\n\nConclusionWe develop two useful web tools to design target sequence (BE-Designer) and to analyze NGS data from experimental results (BE-Analyzer) for CRISPR base editors.

bioinformatics↗

bioRxiv2018

Engrafted parenchymal brain macrophages differ from host microglia in transcriptome, epigenome and responsiveness to challenge

Microglia are yolk sac-derived macrophages residing in the parenchyma of brain and spinal cord, where they interact with neurons and other glial cells by constantly probing their surroundings with dynamic extensions. Following different conditioning paradigms and bone marrow (BM) / hematopoietic stem cell (HSC) transplantation, graft-derived cells seed the brain and persistently contribute to the parenchymal brain macrophage compartment. Here we establish that these cells acquire over time microglia characteristics, including ramified morphology, longevity, radio-resistance and clonal expansion. However, even following prolonged CNS residence, transcriptomes and epigenomes of engrafted HSC-derived macrophages remain distinct from yolk sac-derived host microglia. Furthermore, BM graft-derived cells display discrete responses to peripheral endotoxin challenge, as compared to host microglia. Also in human HSC transplant recipients, engrafted cells remain distinct from host microglia, extending our finding to clinical settings. Collectively, our data emphasize the molecular and functional heterogeneity of parenchymal brain macrophages and highlight potential clinical implications for patients treated by HSC gene therapy.

immunology↗

bioRxiv2018

Cohesin interacts with a panoply of splicing factors required for cell cycle progression and genomic organization

The cohesin complex regulates sister chromatid cohesion, chromosome organization, gene expression, and DNA repair. Here we report that endogenous human cohesin interacts with a panoply of splicing factors and RNA binding proteins, including diverse components of the U4/U6.U5 tri-snRNP complex and several splicing factors that are commonly mutated in cancer. The interactions are enhanced during mitosis, and the interacting splicing factors and RNA binding proteins follow the cohesin cycle and prophase pathway of regulated interactions with chromatin. Depletion of cohesin-interacting splicing factors results in stereotyped cell cycle arrests and alterations in genomic organization. These data support the hypothesis that splicing factors and RNA binding proteins control cell cycle progression and genomic organization via regulated interactions with cohesin and chromatin.\n\nOne Sentence SummaryEndogenous tagging reveals that cohesin interacts with diverse chromatin-bound splicing factors that regulate cell cycle progression and genomic organization in human cells.

cell biology↗

bioRxiv2018

Pseudomonas syringae evades phagocytosis in animal cells through type III effector-mediated inhibition of the LIM kinase-cofilin system

Certain animal and plant pathogenic bacteria have developed virulence factors (including effector proteins) that enable them to overcome host immunity. A plant pathogen, Pseudomonas syringae pv. tomato (Pto), secretes a large repertoire of effectors into plant cells via a type III secretory apparatus, thereby suppressing plant immunity. Here, we show that exposure to Pto caused sepsis in mice. Surprisingly, the effector HopQ1 disrupted phagocytosis by inhibiting actin rearrangement via a direct interaction with the LIM domain of the animal target protein LIM kinase, a key regulator of actin polymerization. The results provide new insights into cross-kingdom pathogenicity of bacteria. The current studies demonstrate that certain plant pathogenic bacteria such as Pto can be fatal in animals due to cross-kingdom host immune suppression.

microbiology↗

bioRxiv2018

CRISPR Cas9 searches for a protospacer adjacent motif by one-dimensional diffusion

Since its discovery, the CRISPR/Cas9 system has been at the focus of fundamental researchers, genome engineers, and the general public alike. Despite being in the spotlight for several years, aspects of the precise molecular mechanism of Cas9 activity remain ambiguous. We use single-molecule Foerster resonance energy transfer (smFRET) to reveal Cas9 target search mechanism with nanometer sensitivity. We have developed single-molecule assays to monitor transient interactions of Cas9 and DNA in real time. Our study shows that Cas9 interacts with the protospacer adjacent motif (PAM) sequence weakly, yet probing neighboring sequences via lateral diffusion. This dynamic mode of interactions leads to translocation of Cas9 to another PAM nearby and consequently an on-target sequence. We propose a model in which lateral diffusion competes with 3-dimensional diffusion and thus might aid PAM finding and consequently on-target binding.

biophysics↗

bioRxiv2018

Single Cell Observations Show Persister Cells Wake Based on Ribosome Content

Since persister cells survive antibiotic treatments through dormancy and resuscitate to reconstitute infections, it is imperative to determine the rate at which these cells revive. Using two sets of Escherichia coli persister cells, those arising naturally at low levels and those generated at high levels by ceasing transcription via rifampicin pretreatment (shown to be bona fide persisters through seven sets of experiments), we used microscopy of single cells to determine that persisters have low levels of antibiotic-corrupting proteins and that their resuscitation is heterogeneous and includes cells that grow immediately. In all, five phenotypes were found for persister cell resuscitation: (i) immediate division, (ii) immediate elongation followed by division, (iii) immediate elongation but no division, (iv) delayed elongation/division, and (v) no growth. In addition, once cell division begins, the growth rate is that of exponential cells. Critically, the greater the ribosome content, the faster the persister cells resuscitate.

microbiology↗

bioRxiv2017

Directed evolution of CRISPR-Cas9 to increase its specificity

The use of CRISPR-Cas9 as a therapeutic reagent is hampered by its off-target effects. Although rationally designed S. pyogenes Cas9 (SpCas9) variants that display higher specificities than the wild-type SpCas9 protein are available, these attenuated Cas9 variants are often poorly efficient in human cells. Here, we have used a directed evolution approach in E. coli to obtain Sniper-Cas9, which shows high specificities without sacrificing on-target activities in human cells.

biochemistry↗

bioRxiv2017

Viable But Non-Culturable Cells Are Persister Cells

Bacteria have two dormant phenotypes: the viable but non-culturable (VBNC) state and the persister state. Both resting stages arise without mutation and both have been linked to chronic infections; however, persister cells revive rapidly whereas the cell population called VBNC is reported to not resuscitate. Here we investigated the relatedness of the two stress-induced phenotypes at the single-cell level by using transmission electron microscopy and fluorescent microscopy to examine cell morphology and by quantifying cell resuscitation. Using the classic starvation conditions to create VBNC cells, we found that the majority of the remaining Escherichia coli population are spherical, have empty cytosol, and fail to resuscitate; however, some of the spherical cells under these classic VBNC-inducing conditions resuscitate immediately (most probably those with dense cytosol). Critically, all the culturable cells became persister cells within 14 days of starvation. We found that the persister cells initially are rodlike, have clear but limited membrane damage, can resuscitate immediately, and gradually become spherical by aging. After 24 h, only rod-shaped persister cells survive, and all the spherical cells lyse. Both cell populations formed under the VBNC-inducing conditions and the persister cells are metabolically inactive. Therefore, the bacterial population consists of dead cells and persister cells in the VBNC-inducing conditions; i.e., the non-lysed particles that do not resuscitate are dead, and the dormant cells that resuscitate are persister cells. Hence, \"VBNC\" and \"persister\" describe the same dormant phenotype.

microbiology↗

bioRxiv2017

Questioning unexpected CRISPR off-target mutations in vivo

To the Editor To the Editor References Recently, Schaefer et al.1 reported that whole genome sequencing (WGS) of two Cas9-treated, gene-corrected mice and a wild-type control mouse unveiled 1,397 single-nucleotide variations (SNVs) and 117 small insertions and deletions (indels) present commonly in the two Cas9-treated mice \"but absent in the uncorrected control\" and from a database of mouse SNVs and indels. There was essentially no sequence homology between the on-target site and these SNVs and indel sites, most of which lacked a protospacer-adjacent motif (PAM) sequence, suggesting that these variations were both small guide RNA (sgRNA)-independent and Cas9-independent, respectively. Nevertheless, the authors made a bold claim that these variations were caused by CRISPR-Cas9 without validating these unexpected off-target effects even at a single SNV or indel site by ...

genomics↗