Clustered, regularly interspaced, short palindromic repeats (CRISPR)/ CRISPR-associated (Cas) systems protect bacteria and archaea from infection by viruses and plasmids. Central to this defense is a ribonucleoprotein complex that produces RNA-guided cleavage of foreign nucleic acids. In DNA-targeting CRISPR-Cas systems, the RNA component of the complex encodes target recognition by forming a sitespecific hybrid (R-loop) with its complement (protospacer) on an invading DNA while displacing the noncomplementary strand. Subsequently, the R-loop structure triggers DNA degradation. Although these reactions have been reconstituted, the exact mechanism of Rloop formation has not been fully resolved. Here, we use singlemolecule DNA supercoiling to directly observe and quantify the dynamics of torque-dependent R-loop formation and dissociation for both Cascade-and Cas9-based CRISPR-Cas systems. We find that the protospacer adjacent motif (PAM) affects primarily the Rloop association rates, whereas protospacer elements distal to the PAM affect primarily R-loop stability. Furthermore, Cascade has higher torque stability than Cas9 by using a conformational locking step. Our data provide direct evidence for directional R-loop formation, starting from PAM recognition and expanding toward the distal protospacer end. Moreover, we introduce DNA supercoiling as a quantitative tool to explore the sequence requirements and promiscuities of orthogonal CRISPR-Cas systems in rapidly emerging gene-targeting applications.magnetic tweezers | genome engineering | crRNA C lustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems constitute an adaptable immune system that protects bacteria and archaea against foreign nucleic acids. The defense is initiated by a ribonucleoprotein (RNP) complex that mediates cleavage of dsDNA (1) or RNA (2, 3). The RNA component (crRNA) of the complex is derived by transcription and posttranscriptional processing from a locus containing CRISPRs (2, 4, 5) in which short spacer fragments were integrated from foreign nucleic acids (6-8). Each transcribed crRNA spacer sequence encodes the recognition of the targets. In DNA-targeting CRISPR-Cas systems, the crRNAs form a hybrid with a matching complement (protospacer) on an invading DNA, which leads to the displacement of the noncomplementary strand. The resulting structure is called an R-loop and constitutes the signal for subsequent DNA degradation. R-loop formation is additionally dependent on a short protospacer adjacent motif (PAM) (Fig. 1A), which provides discrimination between self and nonself DNA in CRISPR systems; it is absolutely required for recognition of the invading DNA but is absent from the host CRISPR array (9).On the basis of sequence homology, different CRISPR-Cas families have been identified (10). We investigate here a type IE and a type II system from Streptococcus thermophilus St-CRISPR4 and St-CRISPR3, respectively. The Cas proteins of type IE systems (4, 11, 12) associate with a crRNA into a mult...
CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against foreign nucleic acids. In type I CRISPR-Cas systems, invading DNA is detected by a large ribonucleoprotein surveillance complex called Cascade. The crRNA component of Cascade is used to recognize target sites in foreign DNA (protospacers) by formation of an R-loop driven by base-pairing complementarity. Using single-molecule supercoiling experiments with near base-pair resolution, we probe here the mechanism of R-loop formation and detect short-lived R-loop intermediates on off-target sites bearing single mismatches. We show that R-loops propagate directionally starting from the protospacer-adjacent motif (PAM). Upon reaching a mismatch, R-loop propagation stalls and collapses in a length-dependent manner. This unambiguously demonstrates that directional zipping of the R-loop accomplishes efficient target recognition by rapidly rejecting binding to off-target sites with PAM-proximal mutations. R-loops that reach the protospacer end become locked to license DNA degradation by the auxiliary Cas3 nuclease/helicase without further target verification.
Diagnosis marks the beginning of any successful therapy. Because many medical conditions progress asymptomatically over extended periods of time, their timely diagnosis remains difficult, and this adversely affects patient prognosis. Focusing on hypercalcemia associated with cancer, we aimed to develop a synthetic biology-inspired biomedical tattoo using engineered cells that would (i) monitor long-term blood calcium concentration, (ii) detect onset of mild hypercalcemia, and (iii) respond via subcutaneous accumulation of the black pigment melanin to form a visible tattoo. For this purpose, we designed cells containing an ectopically expressed calcium-sensing receptor rewired to a synthetic signaling cascade that activates expression of transgenic tyrosinase, which produces melanin in response to persistently increased blood Ca 2+. We confirmed that the melanin-generated color change produced by this biomedical tattoo could be detected with the naked eye and optically quantified. The system was validated in wild-type mice bearing subcutaneously implanted encapsulated engineered cells. All animals inoculated with hypercalcemic breast and colon adenocarcinoma cells developed tattoos, whereas no tattoos were seen in animals inoculated with normocalcemic tumor cells. All tumor-bearing animals remained asymptomatic throughout the 38-day experimental period. Although hypercalcemia is also associated with other pathologies, our findings demonstrate that it is possible to detect hypercalcemia associated with cancer in murine models using this cell-based diagnostic strategy.
Significance The endoplasmic reticulum (ER) and the microtubule (MT) cytoskeleton form a coextensive, dynamic system that pervades eukaryotic cells. The shape of the ER is generated by a set of evolutionarily conserved membrane proteins that are able to control ER morphology and dynamics independently of MTs. Here we uncover that the molecular machinery that determines ER network dynamics can influence the subcellular distribution of MTs. We show that active control of local ER tubule junction density by ER tethering and fusion is important for the spatial organization of the combined ER–MT system. Our work suggests that cells might alter ER junction dynamics to drive formation of MT bundles, which are important structures, e.g., in migrating cells or in neuronal axons.
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