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Cas9-Primed Adaptive Immunity During the CRISPR-Cas Response
Get PDFProkaryotes have developed numerous defense strategies to combat the constant threat of viruses (bacteriophages) that endanger them. Clustered, regularly interspaced short palindromic repeats (CRISPR) loci provide archaea and bacteria with adaptive immune systems that allow them to counteract these rapidly evolving genetic parasites. These diverse systems all generally contain two components: a set of CRISPR-associated (cas) genes and a series of repetitive DNA elements intercalated with variable sequences known as spacers. Following viral infection, these sequences are acquired from the viral genome and integrated in the CRISPR array as new spacers. Spacers are then transcribed into CRISPR RNAs (crRNAs) that direct the Cas nucleases to destroy the invader following sequence-specific recognition of either DNA or RNA. Thus, spacers function as a form of immunologic memory that can be called upon again and again to defend the cell from reinfection. In type II CRISPR-Cas systems, spacer sequences direct the Cas9 nuclease to target infecting bacteriophages and cleave their double-stranded (ds)DNA genomes. Whether and how pre-exiting anti-viral spacers in type II systems affect memory generation and the acquisition of new spacers is unknown. Here, in my thesis work, I demonstrate that previously acquired spacers promote additional spacer capture from the vicinity of the Cas9 cut site at an enhanced rate. I go on to show that Cas9-mediated dsDNA break (DSB) formation is required for spacer-mediated spacer acquisition and that the rate of spacer acquisition is correlated with the efficiency of Cas9 cleavage. As a result of this mechanism, cells with preexisting viral immunity can utilize their spacerderived crRNAs to direct the acquisition of additional spacers in a new phase of immune response known as primed spacer acquisition or priming. A consequence of priming is that immune cells can acquire additional spacers as Cas9 destroys the infecting virus. I go on to show that spacers acquired during Cas9- mediated priming endow potent benefits to bacterial communities faced with virulent bacteriophages. In particular, priming suppresses the emergence of CRISPR escaper and related viruses that emerge during Cas9 targeting. I show that this anti-viral immunity is achieved in three ways. Firstly, priming expands the hosts immune repertoire, thereby improving the existing anti-phage immunity. In addition, I show that primed spacer acquisition allows the host to contain the propagation escapers that have mutations in their target sequence that abrogate Cas9 targeting. Finally, by preemptively immunizing the host with additional spacers during the initial Cas9 targeting response, priming allows the host to anticipate secondary infections by escaper and related viruses. This prophylactic immunity is a unique feature in CRISPR systems that allows type II systems to overcome future threats from viruses that would have overcome the defense provided by the initial anti-viral spacer. CRISPR-Cas immune systems allow their host to rapidly adapt to the viruses that challenge them. Collectively, my thesis work has revealed a new phases of the type II-A CRISPR-Cas9 immune response that is fundamental to how these systems defend their hosts against bacteriophages
What Is Life? Five Great Ideas In Biology
No full texthttps://digitalcommons.rockefeller.edu/book_talk_sir_paul_nurse/1000/thumbnail.jp
CD8+ T Cell Proliferation
No full textSlide 4-18: CD8+ T Cell Proliferation and IFN-γ production after α-human DEC-gag p24 targeting in vitrohttps://digitalcommons.rockefeller.edu/endocytosis/1016/thumbnail.jp
CD1d-EL4/GalCer
No full textSlide 5-20https://digitalcommons.rockefeller.edu/nkt-cells/1019/thumbnail.jp
B16 Melanoma Cells
No full textSlide 5-14: B16 melanoma cells, coated with α-Galactosyl ceramide and injected intravenously, allow many mice to resist B16 s.c.https://digitalcommons.rockefeller.edu/nkt-cells/1012/thumbnail.jp
NKT Cells as Adjuncts to Tumor Immunity
No full textSlide 5-40https://digitalcommons.rockefeller.edu/nkt-cells/1038/thumbnail.jp
Lipmann, Fritz A.
No full textFritz Lipmann, circa 1950s. Photograph by John Brook
Courtesy of the Rockefeller Archive Center
Fritz Albert Lipmann (1899 - 1986) was a German-American biochemist and a co-discoverer in 1945 of coenzyme A. For this, together with other research on coenzyme A, he was awarded the Nobel Prize in Physiology or Medicine in 1953.
See also Deciphering the Pathway By Which Food is Converted into Chemical Energy and National Academy of Sciences Biographical Memoirshttps://digitalcommons.rockefeller.edu/faculty-members/1037/thumbnail.jp
Identification of Gene Expression Changes in Sleep Mutants Associated With Reduced Longevity in Drosophila
Get PDFSleep deprivation has become a common problem in modern society, yet the physiological consequences of sleep deprivation remain poorly understood. Sleep disruption has been shown to shorten lifespan in multiple animal species. In my thesis study, I investigated the relationship between sleep and longevity using Drosophila melanogaster. By analyzing the sleep and longevity profiles from a panel of sleep mutants, I discovered a robust positive correlation between daily sleep time and median longevity. This discovery led to my hypothesis that sleep disruption as a result of genetic mutation would elicit certain gene expression changes that ultimately would lead to shortened lifespan. To identify the genes that exhibited altered expression, I profiled the transcriptomes of wild type flies, aged wild type flies, and two sleep mutants insomniac1 (inc1) and wide awakeD2 (wakeD2) with total RNA samples collected every 4 hours for 2 days. Circadian analysis was performed to detect oscillating transcripts in each group, and the results revealed substantial variations in number of oscillating genes. By comparing the experimental groups to the control wild type group, I discovered large scale rhythmicity changes in the experimental groups. Aged wild type flies predominantly showed a gain of rhythmicity, while loss of rhythmicity was more evident in sleep mutants. Furthermore, with the transcriptome data as input, I developed a differential gene expression (DGE) analysis pipeline to select candidate genes that might serve as the connection between sleep disruption and longevity reduction. In the DGE analysis, each experimental group was compared with the control wild type group. Results from each comparison were subsequently intersected to pinpoint genes that were significantly changed in all experimental groups. Ultimately, 15 candidate genes stood out from the analysis. For the scope of this study, I focused on candidate gene Neuropeptide-like precursor 3 (Nplp3), a putative neuropeptide precursor. RNA sequencing results revealed that expression levels of Nplp3 were reduced in sleep mutants and aged wild type animals, compared to control flies. These results were validated by quantitative reverse transcription polymerase chain reaction (RT-qPCR). More importantly, I found that Nplp3 expression was reduced in several sleep mutants in addition to inc1 and wakeD2. Decreased Nplp3 expression resulted in significant shortening in lifespan but did not affect sleep amount. Transgenic fly strains were generated to selectively overexpress either wild type Nplp3 or Nplp3 without signal peptide using Nplp3 specific driver. Overexpression of normal Nplp3 reduced sleep. Longevity results indicated that signal peptide might be important for Nplp3 function. These findings expanded our understandings of the relationship between sleep and longevity and suggested a potential neuropeptide signaling pathway for regulation of longevity by sleep
Dendritic Cells and Antigen-Transporting Crypt Epithelia
No full textSlide 3-3: Dendritic cells(red) on the surface and antigen-transporting crypt epithelia of the oral lymphoid tonsilhttps://digitalcommons.rockefeller.edu/immunodeficiency-disease/1002/thumbnail.jp
Kanako Shimizu and Shin-ichiro Fujii
No full textKanako Shimizu and Shin-ichiro Fujii, n.d.
RIKEN Center for Allergy and Imnunology
RCAI, Yokohama, Japanhttps://digitalcommons.rockefeller.edu/nkt-cells/1002/thumbnail.jp