Repetitive DNA reeling by the Cascade-Cas3 complex in nucleotide unwinding steps

CRISPR-Cas loci provide an RNA-guided adaptive immune system against invading genetic elements. Interference in type I systems relies on the RNA-guided surveillance complex Cascade for target DNA recognition and the trans-acting Cas3 helicase/nuclease protein for target degradation. Even though the biochemistry of CRISPR interference has been largely covered, the biophysics of DNA unwinding and coupling of the helicase and nuclease domains of Cas3 remains elusive. Here we employed single-molecule FRET to probe the helicase activity with a high spatiotemporal resolution. We show that Cas3 remains tightly associated with the target-bound Cascade complex while reeling in the target DNA using a spring-loaded mechanism. This spring-loaded reeling occurs in distinct bursts of three base pairs, that each underlie three successive 1-nt unwinding events. Reeling is highly repetitive, compensating for inefficient nicking activity of the nuclease domain. Our study reveals that the discontinuous helicase properties of Cas3 and its tight interaction with Cascade ensure well controlled degradation of target DNA only.

Download Full-text

Structural basis for inhibition of the type I-F CRISPR–Cas surveillance complex by AcrIF4, AcrIF7 and AcrIF14

Nucleic Acids Research ◽

10.1093/nar/gkaa1199 ◽

2020 ◽

Author(s):

Clinton Gabel ◽

Zhuang Li ◽

Heng Zhang ◽

Leifu Chang

Keyword(s):

Genome Editing ◽

Conformational Changes ◽

Structural Basis ◽

Type I ◽

Structural Detail ◽

Immune Systems ◽

Helical Bundle ◽

Adaptive Immune ◽

Target Dna ◽

Adaptive Immune Systems

Abstract CRISPR–Cas systems are adaptive immune systems in bacteria and archaea to defend against mobile genetic elements (MGEs) and have been repurposed as genome editing tools. Anti-CRISPR (Acr) proteins are produced by MGEs to counteract CRISPR–Cas systems and can be used to regulate genome editing by CRISPR techniques. Here, we report the cryo-EM structures of three type I-F Acr proteins, AcrIF4, AcrIF7 and AcrIF14, bound to the type I-F CRISPR–Cas surveillance complex (the Csy complex) from Pseudomonas aeruginosa. AcrIF4 binds to an unprecedented site on the C-terminal helical bundle of Cas8f subunit, precluding conformational changes required for activation of the Csy complex. AcrIF7 mimics the PAM duplex of target DNA and is bound to the N-terminal DNA vise of Cas8f. Two copies of AcrIF14 bind to the thumb domains of Cas7.4f and Cas7.6f, preventing hybridization between target DNA and the crRNA. Our results reveal structural detail of three AcrIF proteins, each binding to a different site on the Csy complex for inhibiting degradation of MGEs.

Download Full-text

Comprehensive Mining and Characterization of CRISPR-Cas Systems in Bifidobacterium

Microorganisms ◽

10.3390/microorganisms8050720 ◽

2020 ◽

Vol 8 (5) ◽

pp. 720 ◽

Cited By ~ 3

Author(s):

Meichen Pan ◽

Matthew A. Nethery ◽

Claudio Hidalgo-Cantabrana ◽

Rodolphe Barrangou

Keyword(s):

Genome Engineering ◽

Rare Variants ◽

Adaptive Immune System ◽

Effector Proteins ◽

Type I ◽

Human Gut ◽

Adaptive Immune ◽

Naturally Occurring ◽

History Of

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated cas) systems constitute the adaptive immune system in prokaryotes, which provides resistance against bacteriophages and invasive genetic elements. The landscape of applications in bacteria and eukaryotes relies on a few Cas effector proteins that have been characterized in detail. However, there is a lack of comprehensive studies on naturally occurring CRISPR-Cas systems in beneficial bacteria, such as human gut commensal Bifidobacterium species. In this study, we mined 954 publicly available Bifidobacterium genomes and identified CRIPSR-Cas systems in 57% of these strains. A total of five CRISPR-Cas subtypes were identified as follows: Type I-E, I-C, I-G, II-A, and II-C. Among the subtypes, Type I-C was the most abundant (23%). We further characterized the CRISPR RNA (crRNA), tracrRNA, and PAM sequences to provide a molecular basis for the development of new genome editing tools for a variety of applications. Moreover, we investigated the evolutionary history of certain Bifidobacterium strains through visualization of acquired spacer sequences and demonstrated how these hypervariable CRISPR regions can be used as genotyping markers. This extensive characterization will enable the repurposing of endogenous CRISPR-Cas systems in Bifidobacteria for genome engineering, transcriptional regulation, genotyping, and screening of rare variants.

Download Full-text

Defense Against Viral Attack: Single-Molecule View on a Bacterial Adaptive Immune System

Biophysical Journal ◽

10.1016/j.bpj.2013.11.178 ◽

2014 ◽

Vol 106 (2) ◽

pp. 22a

Author(s):

Timothy Blosser ◽

Edze Westra ◽

Luuk Loeff ◽

Cees Dekker ◽

Stan Brouns ◽

...

Keyword(s):

Immune System ◽

Single Molecule ◽

Adaptive Immune System ◽

Adaptive Immune

Download Full-text

Defective innate immunity predisposes murine neonates to poor sepsis outcome but is reversed by TLR agonists

Blood ◽

10.1182/blood-2008-01-130500 ◽

2008 ◽

Vol 112 (5) ◽

pp. 1750-1758 ◽

Cited By ~ 117

Author(s):

James L. Wynn ◽

Philip O. Scumpia ◽

Robert D. Winfield ◽

Matthew J. Delano ◽

Kindra Kelly-Scumpia ◽

...

Keyword(s):

Innate Immunity ◽

Immune System ◽

Adaptive Immune System ◽

Neutrophil Recruitment ◽

Polymicrobial Sepsis ◽

Type I ◽

Sepsis Mortality ◽

Tlr Agonists ◽

Adaptive Immune ◽

Increased Risk

Abstract Neonates exhibit an increased risk of sepsis mortality compared with adults. We show that in contrast to adults, survival from polymicrobial sepsis in murine neonates does not depend on an intact adaptive immune system and is not improved by T cell–directed adaptive immunotherapy. Furthermore, neonates manifest an attenuated inflammatory and innate response to sepsis, and have functional defects in their peritoneal CD11b+ cells. Activation of innate immunity with either a Toll-like receptor 4 (TLR4) or TLR7/8 agonist, but not a TLR3 agonist, increased the magnitude, but abbreviated the early systemic inflammatory response, reduced bacteremia, and improved survival to polymicrobial sepsis. TLR4 agonist pretreatment enhanced peritoneal neutrophil recruitment with increased oxidative burst production, whereas the TLR7/8 agonist also enhanced peritoneal neutrophil recruitment with increased phagocytic ability. These benefits were independent of the adaptive immune system and type I interferon signaling. Improving innate immune function with select TLR agonists may be a useful strategy to prevent neonatal sepsis mortality.

Download Full-text

SLPI and elafin: one glove, many fingers

Clinical Science ◽

10.1042/cs20050115 ◽

2005 ◽

Vol 110 (1) ◽

pp. 21-35 ◽

Cited By ~ 174

Author(s):

Steven E. Williams ◽

Thomas I. Brown ◽

Ali Roghanian ◽

Jean-Michel Sallenave

Keyword(s):

Immune Response ◽

Immune System ◽

Immune Responses ◽

Adaptive Immune Response ◽

Adaptive Immune System ◽

Innate Immune ◽

Type I ◽

Adaptive Immune ◽

Pathological Conditions ◽

Related Proteins

Elafin and SLPI (secretory leucocyte protease inhibitor) have multiple important roles both in normal homoeostasis and at sites of inflammation. These include antiprotease and antimicrobial activity as well as modulation of the response to LPS (lipopolysaccharide) stimulation. Elafin and SLPI are members of larger families of proteins secreted predominantly at mucosal sites, and have been shown to be modulated in multiple pathological conditions. We believe that elafin and SLPI are important molecules in the controlled functioning of the innate immune system, and may have further importance in the integration of this system with the adaptive immune response. Recent interest has focused on the influence of inflamed tissues on the recruitment and phenotypic modulation of cells of the adaptive immune system and, indeed, the local production of elafin and SLPI indicate that they are ideally placed in this regard. Functionally related proteins, such as the defensins and cathelicidins, have been shown to have direct effects upon dendritic cells with potential alteration of their phenotype towards type I or II immune responses. This review addresses the multiple functions of elafin and SLPI in the inflammatory response and discusses further their roles in the development of the adaptive immune response.

Download Full-text

Priming in a permissive type I-C CRISPR-Cas system reveals distinct dynamics of spacer acquisition and loss

10.1101/137067 ◽

2017 ◽

Author(s):

Chitong Rao ◽

Denny Chin ◽

Alexander W. Ensminger

Keyword(s):

Legionella Pneumophila ◽

Adaptive Immune System ◽

Type I ◽

Adaptive Immune ◽

Protospacer Adjacent Motif ◽

Wide Range ◽

Sequence Complementarity ◽

Bona Fide ◽

Dramatic Shift ◽

Sequence Constraints

AbstractCRISPR-Cas is a bacterial and archaeal adaptive immune system that uses short, invader-derived sequences termed spacers to target invasive nucleic acids. Upon recognition of previously encountered invaders, the system can stimulate secondary spacer acquisitions, a process known as primed adaptation. Previous studies of primed adaptation have been complicated by intrinsically high interference efficiency of most systems against bona fide targets. As such, most primed adaptation to date has been studied within the context of imperfect sequence complementarity between spacers and targets. Here, we take advantage of a native type I-C CRISPR-Cas system in Legionella pneumophila that displays robust primed adaptation even within the context of a perfectly matched target. Using next-generation sequencing to survey acquired spacers, we observe strand bias and positional preference that are consistent with a 3′ to 5′ translocation of the adaptation machinery. We show that spacer acquisition happens in a wide range of frequencies across the plasmid, including a remarkable hotspot that predominates irrespective of the priming strand. We systematically characterize protospacer sequence constraints in both adaptation and interference and reveal extensive flexibilities regarding the protospacer adjacent motif in both processes. Lastly, in a strain with a genetically truncated CRISPR array, we observe greatly increased interference efficiency coupled with a dramatic shift away from spacer acquisition towards spacer loss. Based on these observations, we propose that the Legionella type I-C system represents a powerful model to study primed adaptation and the interplay between CRISPR interference and adaptation.

Download Full-text

Real-time observation of flexible domain movements in Cas9

10.1101/122069 ◽

2017 ◽

Cited By ~ 3

Author(s):

Saki Osuka ◽

Kazushi Isomura ◽

Shohei Kajimoto ◽

Tomotaka Komori ◽

Hiroshi Nishimasu ◽

...

Keyword(s):

Single Molecule ◽

Dna Cleavage ◽

Potential Role ◽

Domain Architecture ◽

Guide Rna ◽

Single Molecule Fret ◽

Target Dna ◽

Time Observation ◽

Real Time Observation

ABSTRACTThe CRISPR-associated protein Cas9 is a widely used genome editing tool that recognizes and cleaves target DNA through the assistance of a single-guide RNA (sgRNA). Structural studies have demonstrated the multi-domain architecture of Cas9 and sequential domain movements upon binding to the sgRNA and the target DNA. These studies also hinted at the flexibility between domains, but whether these flexible movements occur in solution is unclear. Here, we directly observed dynamic fluctuations of multiple Cas9 domains, using single-molecule FRET. The flexible domain movements allow Cas9 to adopt transient conformations beyond those captured in the crystal structures. Importantly, the HNH nuclease domain in Cas9 only accessed the DNA cleavage position during such flexible movements, suggesting the importance of this flexibility in the DNA cleavage process. Our FRET data also revealed the conformational flexibility of apo-Cas9, which may play a role in the assembly with the sgRNA. Collectively, our results highlight the potential role of domain fluctuations in driving Cas9-catalyzed DNA cleavage.

Download Full-text

CRISPR-Cas: biology, mechanisms and relevance

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2015.0496 ◽

2016 ◽

Vol 371 (1707) ◽

pp. 20150496 ◽

Cited By ~ 100

Author(s):

Frank Hille ◽

Emmanuelle Charpentier

Keyword(s):

Dna Sequences ◽

Adaptive Immune System ◽

Crispr Locus ◽

Biochemical Processes ◽

Adaptive Immune ◽

Physiological Properties ◽

Target Dna ◽

Associated Proteins ◽

Cas Proteins

Prokaryotes have evolved several defence mechanisms to protect themselves from viral predators. Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) display a prokaryotic adaptive immune system that memorizes previous infections by integrating short sequences of invading genomes—termed spacers—into the CRISPR locus. The spacers interspaced with repeats are expressed as small guide CRISPR RNAs (crRNAs) that are employed by Cas proteins to target invaders sequence-specifically upon a reoccurring infection. The ability of the minimal CRISPR-Cas9 system to target DNA sequences using programmable RNAs has opened new avenues in genome editing in a broad range of cells and organisms with high potential in therapeutical applications. While numerous scientific studies have shed light on the biochemical processes behind CRISPR-Cas systems, several aspects of the immunity steps, however, still lack sufficient understanding. This review summarizes major discoveries in the CRISPR-Cas field, discusses the role of CRISPR-Cas in prokaryotic immunity and other physiological properties, and describes applications of the system as a DNA editing technology and antimicrobial agent. This article is part of the themed issue ‘The new bacteriology’.

Download Full-text

Expression of Toll-Like Receptors on Peripheral Blood Cells After Allogeneic Stem Cell Transplantation: Results of a Prospective Study,

Blood ◽

10.1182/blood.v118.21.4071.4071 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4071-4071

Author(s):

Cristina Skert ◽

Manuela Fogli ◽

Simone Perucca ◽

Simona Fiorentini ◽

Emirena Garrafa ◽

...

Keyword(s):

Stem Cell ◽

Immune System ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Allogeneic Stem Cell Transplantation ◽

Acute Gvhd ◽

Adaptive Immune System ◽

Type I ◽

Ifn Gamma ◽

Adaptive Immune

Abstract Abstract 4071 Introduction. Emerging trends emphasize the importance of both innate and adaptive immune system in the response against infections, and in the pathogenesis of autoimmune and graft-versus-host (GVHD) diseases. Pattern recognition receptors such as Toll-like receptors (TLRs) play a key role in the cross-talk between innate and adaptive immune system. TLRs belong to type I transmembrane glycoprotein receptor family and recognize pathogen-associated molecular patterns (PAMPs), such as common protein, carbohydrate or DNA/RNA pattern motifs. TLRs are also receptors for endogenous ligands and damaged tissue, suggesting that both pathogen-derived molecules and products of damaged tissue can trigger signals which are responsible for the regulation of innate and adaptive immune responses. Extracellular ligands are recognized by surface TLRs (TLR1,TLR2,TLR4,TLR5, and TLR6). Intracellular TLRs (TLR3,TLR7,TLR8 and TLR9) bind mainly to foreign nucleic acids and sometimes detect self DNA/RNA. Aim of the study. Very little is known about expression and function of TLRs in vivo in patients who underwent allogeneic stem cell transplantation (SCT). The aim of this study was to evaluate the expression of TLRs on lymphocytes and monocytes in relation to the onset of acute GVHD. Methods. The expression of TLRs on lymphocytes and monocytes was analysed by flow cytometry as mean fluorescence intensity at day +30 and at the onset of GVHD. Functional data were obtained by ELISA assay after TLRs activation. The cell supernatants were collected and assayed for TNF-alpha, IFN-gamma and MCP-1. Relative induction of these cytokines was calculated in relation with unstimulated controls. Results. We analyzed 17 healthy donors and 34 patients. Median age was 46 years (range, 22–64) and 22 patients were male. Acute GVHD developed in 19 patients (12 with grade >=2). Clinical and transplant characteristics did not differ in patients with and without GVHD. Lymphocytes and monocytes of patients with acute GVHD showed higher levels of TLR5 (3,5±2,3 vs1,9±1,6 p=0,03; 25,8±25,9 vs 9,0±5,0 p=0,02) and a decreased expression of TLR1 (2,5±2,8 vs 4,3±2,8 p=0,02; 21,4±21,9 vs 54,9±37,4 p=0,005) and TLR9 (63,8±30,4 vs 111,1±62,9 p=0,03; 85,3±73,9 vs 164,2±90,6 p=0,01). IFN-gamma relative induction post-stimulation of TLR2,3,4 and 9 was significantly decreased in patients with acute GVHD (p< 0,04). Conclusions. TLRs show a different profile of expression in patients with acute GVHD in comparison with patients without it. These results suggest that the innate immune response via TLRs activation could be involved in the development of GVHD. In particular, a decreased expression of TLR-9 (receptor of hypomethylated DNA) on lymphocytes and monocytes can promote TLR-7 activation, inducing type I interferons and other pro-inflammatory cytokines. TLR-1 and −5, which are ligands for bacterial cell wall, could also be involved in the pathogenesis of GVHD. Moreover, acute GVHD negatively correlates with IFN-gamma production upon TLR2,3,4 and 9 activation. The assessment of a larger number of patients could be useful to understand the complex interplay among pathogens, self or non-self DNA and RNA, and the immune system. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding

Nucleic Acids Research ◽

10.1093/nar/gkz1058 ◽

2019 ◽

Cited By ~ 2

Author(s):

Ikenna C Okafor ◽

Digvijay Singh ◽

Yanbo Wang ◽

Minhee Jung ◽

Haobo Wang ◽

...

Keyword(s):

Single Molecule ◽

Genome Engineering ◽

Dna Unwinding ◽

Dependent Manner ◽

Guide Rnas ◽

Single Molecule Fret ◽

Wide Range ◽

The Cost ◽

Single Molecule Analysis ◽

Target Activity

Abstract Cas9 has made a wide range of genomic manipulation possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity, but at the cost of the on-target activity. DNA unwinding is a checkpoint before cleavage by Cas9, and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in engineered Cas9s. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used, but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two non-matching guanines added to the 5′ end were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5′ end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering.

Download Full-text