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PNAS -- Science Sessions Podcasts

PNAS -- Science Sessions Podcasts | SynBioFromLeukipposInstitute | Scoop.it
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Podcast: James Collins explains how researchers can rewire bacterial cells and control multiple genes simultaneously within a single cell http://bit.ly/1lANPC2

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Droplet microfluidics for synthetic biology - Lab on a Chip 

Droplet microfluidics for synthetic biology - Lab on a Chip  | SynBioFromLeukipposInstitute | Scoop.it
Synthetic biology is an interdisciplinary field that aims to engineer biological systems for useful purposes. Organism engineering often requires the optimization of individual genes and/or entire biological pathways (consisting of multiple genes). Advances in DNA sequencing and synthesis have recently begun to enable the possibility of evaluating thousands of gene variants and hundreds of thousands of gene combinations. However, such large-scale optimization experiments remain cost-prohibitive to researchers following traditional molecular biology practices, which are frequently labor-intensive and suffer from poor reproducibility. Liquid handling robotics may reduce labor and improve reproducibility, but are themselves expensive and thus inaccessible to most researchers. Microfluidic platforms offer a lower entry price point alternative to robotics, and maintain high throughput and reproducibility while further reducing operating costs through diminished reagent volume requirements. Droplet microfluidics have shown exceptional promise for synthetic biology experiments, including DNA assembly, transformation/transfection, culturing, cell sorting, phenotypic assays, artificial cells and genetic circuits.
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Connecting Biology to Electronics: Molecular Communication via Redox Modality

Biology and electronics are both expert at for accessing, analyzing, and responding to information. Biology uses ions, small molecules, and macromolecules to receive, analyze, store, and transmit information, whereas electronic devices receive input in the form of electromagnetic radiation, process the information using electrons, and then transmit output as electromagnetic waves. Generating the capabilities to connect biology–electronic modalities offers exciting opportunities to shape the future of biosensors, point-of-care medicine, and wearable/implantable devices. Redox reactions offer unique opportunities for bio-device communication that spans the molecular modalities of biology and electrical modality of devices. Here, an approach to search for redox information through an interactive electrochemical probing that is analogous to sonar is adopted. The capabilities of this approach to access global chemical information as well as information of specific redox-active chemical entities are illustrated using recent examples. An example of the use of synthetic biology to recognize external molecular information, process this information through intracellular signal transduction pathways, and generate output responses that can be detected by electrical modalities is also provided. Finally, exciting results in the use of redox reactions to actuate biology are provided to illustrate that synthetic biology offers the potential to guide biological response through electrical cues.
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Autodesk Gallery Exhibits | Bio Computation and Aerospace

Autodesk Gallery Exhibits | Bio Computation and Aerospace | SynBioFromLeukipposInstitute | Scoop.it
The Airbus concept plane uses synthetic biology, embodying what air transport could look like in 2050.
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Synthetic biology – Engineering cell-based biomedical devices

Synthetic biology – Engineering cell-based biomedical devices | SynBioFromLeukipposInstitute | Scoop.it
Synthetic biology applies rational bottom-up engineering principles to create cell-based biological systems with novel and enhanced functionality to address currently unmet clinical needs. In this review, we provide a brief overview of the state-of-the-art in cell-based therapeutic solutions, focusing on how these integrated biological devices can enhance and complement the natural functionality of cells in order to provide novel treatments. We also highlight some blueprints for synthetic biology-inspired approaches to developing cell-based cancer therapies, and briefly discuss their future clinical potential.
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Genetic Constructor: An online DNA design platform

Genetic Constructor is a cloud Computer Aided Design (CAD) application developed to support synthetic biologists from design intent through DNA fabrication and experiment iteration. The platform allows users to design, manage, and navigate complex DNA constructs and libraries, using a new visual language that focuses on functional parts abstracted from sequence. Features like combinatorial libraries and automated primer design allow the user to separate design from construction by focusing on functional intent, and design constraints aid iterative refinement of designs. A plugin architecture enables contributions from scientists and coders to leverage existing powerful software and connect to DNA foundries. The software is easily accessible and platform agnostic, free for academics, and available in an open-source community edition. Genetic Constructor seeks to democratize DNA design, manufacture, and access to tools and services from the synthetic biology community.
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Engineers develop a programmable 'camouflaging' material inspired by octopus skin

Engineers develop a programmable 'camouflaging' material inspired by octopus skin | SynBioFromLeukipposInstitute | Scoop.it
For the octopus and cuttlefish, instantaneously changing their skin color and pattern to disappear into the environment is just part of their camouflage prowess. These animals can also swiftly and reversibly morph their ski
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RNA targeting with CRISPR–Cas13

RNA targeting with CRISPR–Cas13 | SynBioFromLeukipposInstitute | Scoop.it
RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference1, 2, 3 can efficiently knockdown RNAs, but it is prone to off-target effects4, and visualizing RNAs typically relies on the introduction of exogenous tags5. Here we demonstrate that the class 2 type VI6, 7 RNA-guided RNA-targeting CRISPR–Cas effector Cas13a8 (previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of 15 orthologues, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective in an interference assay in Escherichia coli. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference and improved specificity. Catalytically inactive LwaCas13a maintains targeted RNA binding activity, which we leveraged for programmable tracking of transcripts in live cells. Our results establish CRISPR–Cas13a as a flexible platform for studying RNA in mammalian cells and therapeutic development.
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Meet the scientist leading the charge to bring morality to genetic engineering

Meet the scientist leading the charge to bring morality to genetic engineering | SynBioFromLeukipposInstitute | Scoop.it
To avoid missteps with a powerful genetic engineering technology that can modify species, one scientist demands total transparency.
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Microbes Might Paint Your Next Party Dress

Microbes Might Paint Your Next Party Dress | SynBioFromLeukipposInstitute | Scoop.it
The official “fashion month,” September has concluded its parade of gorgeous outfits. These contain harmful dyes, though. Can microbes make safer colors?
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Return of Analog Computing for Cell Simulation

Return of Analog Computing for Cell Simulation | SynBioFromLeukipposInstitute | Scoop.it
Return of Analog Computing for Cell Simulation http://evolving-science.com/bioengineering-computational-synthetic-biology/return-analog-computing-cell-simulation-00135
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Engineering the Genetic Code in Cells and Animals: Biological Considerations and Impacts

Expansion of the genetic code allows unnatural amino acids (Uaas) to be site-specifically incorporated into proteins in live biological systems, thus enabling novel properties selectively introduced into target proteins in vivo for basic biological studies and for engineering of novel biological functions. Orthogonal components including tRNA and aminoacyl-tRNA synthetase (aaRS) are expressed in live cells to decode a unique codon (often the amber stop codon UAG) as the desired Uaa. Initially developed in E. coli, this methodology has now been expanded in multiple eukaryotic cells and animals. In this Account, we focus on addressing various biological challenges for rewriting the genetic code, describing impacts of code expansion on cell physiology and discussing implications for fundamental studies of code evolution. Specifically, a general method using the type-3 polymerase III promoter was developed to efficiently express prokaryotic tRNAs as orthogonal tRNAs and a transfer strategy was devised to generate Uaa-specific aaRS for use in eukaryotic cells and animals. The aaRSs have been found to be highly amenable for engineering substrate specificity toward Uaas that are structurally far deviating from the native amino acid, dramatically increasing the stereochemical diversity of Uaas accessible. Preparation of the Uaa in ester or dipeptide format markedly increases the bioavailability of Uaas to cells and animals. Nonsense-mediated mRNA decay (NMD), an mRNA surveillance mechanism of eukaryotic cells, degrades mRNA containing a premature stop codon. Inhibition of NMD increases Uaa incorporation efficiency in yeast and Caenorhabditis elegans. In bacteria, release factor one (RF1) competes with the orthogonal tRNA for the amber stop codon to terminate protein translation, leading to low Uaa incorporation efficiency. Contradictory to the paradigm that RF1 is essential, it is discovered that RF1 is actually nonessential in E. coli. Knockout of RF1 dramatically increases Uaa incorporation efficiency and enables Uaa incorporation at multiple sites, making it feasible to use Uaa for directed evolution. Using these strategies, the genetic code has been effectively expanded in yeast, mammalian cells, stem cells, worms, fruit flies, zebrafish, and mice. It is also intriguing to find out that the legitimate UAG codons terminating endogenous genes are not efficiently suppressed by the orthogonal tRNA/aaRS in E. coli. Moreover, E. coli responds to amber suppression pressure promptly using transposon insertion to inactivate the introduced orthogonal aaRS. Persistent amber suppression evading transposon inactivation leads to global proteomic changes with a notable up-regulation of a previously uncharacterized protein YdiI, for which an unexpected function of expelling plasmids is discovered. Genome integration of the orthogonal tRNA/aaRS in mice results in minor changes in RNA transcripts but no significant physiological impairment. Lastly, the RF1 knockout E. coli strains afford a previously unavailable model organism for studying otherwise intractable questions on code evolution in real time in the laboratory. We expect that genetically encoding Uaas in live systems will continue to unfold new questions and directions for studying biology in vivo, investigating the code itself, and reprograming genomes for synthetic biology.
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Biosensors detect harmful bugs in the lungs of cystic fibrosis patients

Biosensors detect harmful bugs in the lungs of cystic fibrosis patients | SynBioFromLeukipposInstitute | Scoop.it
A team of Imperial researchers has developed a tool which 'lights up' when it detects the chemical signature of harmful bacteria in the lung. In a clinical first, the group from the Department of Medicine used the tools, called cell-free biosensors, to test samples of sputum (phlegm) from patients...
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Expanding and reprogramming the genetic code

Nature uses a limited, conservative set of amino acids to synthesize proteins. The ability to genetically encode an expanded set of building blocks with new chemical and physical properties is transforming the study, manipulation and evolution of proteins, and is enabling diverse applications, including approaches to probe, image and control protein function, and to precisely engineer therapeutics. Underpinning this transformation are strategies to engineer and rewire translation. Emerging strategies aim to reprogram the genetic code so that noncanonical biopolymers can be synthesized and evolved, and to test the limits of our ability to engineer the translational machinery and systematically recode genomes.
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Gene circuit switches on inside cancer cells, triggers immune attack

Gene circuit switches on inside cancer cells, triggers immune attack | SynBioFromLeukipposInstitute | Scoop.it
Researchers at MIT have developed a synthetic gene circuit that triggers the body’s immune system to attack cancers when it detects signs of the disease.
The circuit, which will only activate a therapeutic response when it detects two specific cancer markers, is described in a paper published today in the journal Cell.
Immunotherapy is widely seen as having considerable potential in the fight against a range of cancers. The approach has been demonstrated successfully in several recent clinical trials, according to Timothy Lu, associate professor of biological engineering and of electrical engineering and computer science at MIT.
“There has been a lot of clinical data recently suggesting that if you can stimulate the immune system in the right way you can get it to recognize cancer,” says Lu, who is head of the Synthetic Biology Group in MIT’s Research Laboratory of Electronics. “Some of the best examples of this are what are called checkpoint inhibitors, where essentially cancers put up stop signs [that prevent] T-cells from killing them. There are antibodies that have been developed now that basically block those inhibitory signals and allow the immune system to act against the cancers.”
However, despite this success, the use of immunotherapy remains limited by the scarcity of tumor-specific antigens — substances that can trigger an immune system response to a particular type of cancer. The toxicity of some therapies, when delivered as a systemic treatment to the whole body, for example, is another obstacle.
What’s more, the treatments are not successful in all cases. Indeed, even in some of the most successful tests, only 30-40 percent of patients will respond to a given therapy, Lu says.
As a result, there is now a push to develop combination therapies, in which different but complementary treatments are used to boost the immune response. So, for example, if one type of immunotherapy is used to knock out an inhibitory signal produced by a cancer, and the tumor responds by upregulating a second signal, an additional therapy could then be used to target this one as well, Lu says.
“Our belief is that there is a need to develop much more specific, targeted immunotherapies that work locally at the tumor site, rather than trying to treat the entire body systemically,” he says. “Secondly, we want to produce multiple immunotherapies from a single package, and therefore be able to stimulate the immune system in multiple different ways.”
To do this, Lu and a team including MIT postdocs Lior Nissim and Ming-Ru Wu, have built a gene circuit encoded in DNA designed to distinguish cancer cells from noncancer cells.
The circuit, which can be customized to respond to different types of tumor, is based on the simple AND gates used in electronics. Such AND gates will only switch on a circuit when two inputs are present.
Cancer cells differ from normal cells in the profile of their gene expression. So the researchers developed synthetic promoters — DNA sequences designed to initiate gene expression but only in cancer cells.
The circuit is delivered to cells in the affected area of the body using a virus. The synthetic promotors are then designed to bind to certain proteins that are active in tumor cells, causing the promoters to turn on.
“Only when two of these cancer promoters are activated, does the circuit itself switch on,” Lu says.
This allows the circuit to target tumors more accurately than existing therapies, as it requires two cancer-specific signals to be present before it will respond.
Once activated, the circuit expresses proteins designed to direct the immune system to target the tumor cells, including surface T cell engagers, which direct T cells to kill the cells. The circuit also expresses a checkpoint inhibitor designed to lift the brakes on T cell activity.
When the researchers tested the circuit in vitro, they found that it was able to detect ovarian cancer cells from amongst other noncancerous ovarian cells and other cell types.
They then tested the circuit in mice implanted with ovarian cancer cells, and demonstrated that it could trigger T cells to seek out and kill the cancer cells without harming other cells around them.
Finally, the researchers showed that the circuit could be readily converted to target other cancer cells.
“We identified other promoters that were selective for breast cancer, and when these were encoded into the circuit, it would target breast cancer cells over other types of cell,” Lu says.
Ultimately, they hope they will also be able to use the system to target other diseases, such as rheumatoid arthritis, inflammatory bowel disease, and other autoimmune diseases.
This advance will open up a new front against cancer, says Martin Fussenegger, a professor of biotechnology and bioengineering at ETH Zurich in Switzerland, who was not involved in the research.
“First author Lior Nissim, who pioneered the very first genetic circuit targeting tumor cells, has now teamed up with Timothy Lu to design RNA-based immunomodulatory gene circuits that take cancer immunotherapy to a new level,” Fussenegger says. “The design of this highly complex tumor-killing gene circuit was made possible by meticulous optimization and integration of several components that target and program tumor cells to become a specific prey for the immune system — this is very smart technology.”
The researchers now plan to test the circuit more fully in a range of cancer models. They are also aiming to develop a delivery system for the circuit, which would be both flexible and simple to manufacture and use.
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Towards dynamic genome-scale models.

The analysis of the dynamic behaviour of genome-scale models of metabolism (GEMs) currently presents considerable challenges because of the difficulties of simulating such large and complex networks. Bacterial GEMs can comprise about 5000 reactions and metabolites, and encode a huge variety of growth conditions; such models cannot be used without sophisticated tool support. This article is intended to aid modellers, both specialist and non-specialist in computerized methods, to identify and apply a suitable combination of tools for the dynamic behaviour analysis of large-scale metabolic designs. We describe a methodology and related workflow based on publicly available tools to profile and analyse whole-genome-scale biochemical models. We use an efficient approximative stochastic simulation method to overcome problems associated with the dynamic simulation of GEMs. In addition, we apply simulative model checking using temporal logic property libraries, clustering and data analysis, over time series of reaction rates and metabolite concentrations. We extend this to consider the evolution of reaction-oriented properties of subnets over time, including dead subnets and functional subsystems. This enables the generation of abstract views of the behaviour of these models, which can be large-up to whole genome in size-and therefore impractical to analyse informally by eye. We demonstrate our methodology by applying it to a reduced model of the whole-genome metabolism of Escherichia coli K-12 under different growth conditions. The overall context of our work is in the area of model-based design methods for metabolic engineering and synthetic biology.
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Autodesk Gallery Exhibits | Digital Tools In Life Sciences

Autodesk Gallery Exhibits | Digital Tools In Life Sciences | SynBioFromLeukipposInstitute | Scoop.it
Digital technology for manufacturing, film, and video games may provide tools to help scientists tackle some of the most pressing challenges to human health.
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Biohacking Resources for Kids

A simple set of resources to get kids or families started in the world of biohacking. Created for Luis Rey and the folks at the Colegio de San Francisco de Paula on the occasion of the Singularity Summit in Sevilla, March 2015.
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Commentary: CRISPR-Cas Encoding of a Digital Movie into the Genomes of a Population of Living Bacteria

Comment on
CRISPR-Cas encoding of a digital movie into the genomes of a population of living bacteria. [Nature. 2017]
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Stretchable surfaces with programmable 3D texture morphing for synthetic camouflaging skins

Some animals, such as cephalopods, use soft tissue to change shape reversibly for camouflage and object manipulation. Pikul et al. used fixed-length fiber mesh embedded in a silicone elastomer to transform a flat object into a 3D structure by inflating membranes (see the Perspective by Laschi). Painted models of rocks and plants were also created that could be morphed to fully blend into their surroundings.
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How switches work in bacteria

How switches work in bacteria | SynBioFromLeukipposInstitute | Scoop.it
Many bacteria have molecular control elements, via which they can switch on and off genes. These riboswitches also open up new options in the development of antibiotics or for the detection and decomposition of environmental toxins. Researchers of Karlsruhe Institute of Technology (KIT), Heidelberg University, and Freie Universität Berlin have now used light optical microscopy of single molecules to fundamentally study the way riboswitches work. This is reported in Nature Chemical Biology. (DOI: 10.1038/nchembio.2476)
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Chemical Neuroscience | The New York Academy of Sciences

Chemical Neuroscience | The New York Academy of Sciences | SynBioFromLeukipposInstitute | Scoop.it
This symposium will explore how chemical biology is being used to increase our understanding of the human brain in health and disease.
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Droplet microfluidics for synthetic biology - Lab on a Chip 

Droplet microfluidics for synthetic biology - Lab on a Chip  | SynBioFromLeukipposInstitute | Scoop.it
Synthetic biology is an interdisciplinary field that aims to engineer biological systems for useful purposes. Organism engineering often requires the optimization of individual genes and/or entire biological pathways (consisting of multiple genes). Advances in DNA sequencing and synthesis have recently begun to ena
Lab on a Chip Recent Review Articles
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Programmable assembly of pressure sensors using pattern-forming bacteria

Programmable assembly of pressure sensors using pattern-forming bacteria | SynBioFromLeukipposInstitute | Scoop.it
Biological systems can generate microstructured materials that combine organic and inorganic components and possess diverse physical and chemical properties. However, these natural processes in materials fabrication are not readily programmable. Here, we use a synthetic-biology approach to assemble patterned materials. We demonstrate programmable fabrication of three-dimensional (3D) materials by printing engineered self-patterning bacteria on permeable membranes that serve as a structural scaffold. Application of gold nanoparticles to the colonies creates hybrid organic-inorganic dome structures. The dynamics of the dome structures' response to pressure is determined by their geometry (colony size, dome height, and pattern), which is easily modified by varying the properties of the membrane (e.g., pore size and hydrophobicity). We generate resettable pressure sensors that process signals in response to varying pressure intensity and duration.
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A Cell-Free Biosensor for Detecting Quorum Sensing Molecules in P. aeruginosa-Infected Respiratory Samples

A Cell-Free Biosensor for Detecting Quorum Sensing Molecules in P. aeruginosa-Infected Respiratory Samples | SynBioFromLeukipposInstitute | Scoop.it
Synthetic biology designed cell-free biosensors are a promising new tool for the detection of clinically relevant biomarkers in infectious diseases. Here, we report that a modular DNA-encoded biosensor in cell-free protein expression systems can be used to measure a bacterial biomarker of Pseudomonas aeruginosa infection from human sputum samples. By optimizing the cell-free system and sample extraction, we demonstrate that the quorum sensing molecule 3-oxo-C12-HSL in sputum samples from cystic fibrosis lungs can be quantitatively measured at nanomolar levels using our cell-free biosensor system, and is comparable to LC–MS measurements of the same samples. This study further illustrates the potential of modular cell-free biosensors as rapid, low-cost detection assays that can inform clinical practice.
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How type II CRISPR–Cas establish immunity through Cas1–Cas2-mediated spacer integration

How type II CRISPR–Cas establish immunity through Cas1–Cas2-mediated spacer integration | SynBioFromLeukipposInstitute | Scoop.it
CRISPR (clustered regularly interspaced short palindromic repeats) and the nearby Cas (CRISPR-associated) operon establish an RNA-based adaptive immunity system in prokaryotes1, 2, 3, 4, 5. Molecular memory is created when a short foreign DNA-derived prespacer is integrated into the CRISPR array as a new spacer6, 7, 8, 9. Whereas the RNA-guided CRISPR interference mechanism varies widely among CRISPR–Cas systems, the spacer integration mechanism is essentially identical7, 8, 9. The conserved Cas1 and Cas2 proteins form an integrase complex consisting of two distal Cas1 dimers bridged by a Cas2 dimer6, 10. The prespacer is bound by Cas1–Cas2 as a dual-forked DNA, and the terminal 3′-OH of each 3′ overhang serves as an attacking nucleophile during integration11, 12, 13, 14. The prespacer is preferentially integrated into the leader-proximal region of the CRISPR array1, 7, 10, 15, guided by the leader sequence and a pair of inverted repeats inside the CRISPR repeat7, 15, 16, 17, 18, 19, 20. Spacer integration in the well-studied Escherichia coli type I–E CRISPR system also relies on the bacterial integration host factor21, 22. In type II–A CRISPR, however, Cas1–Cas2 alone integrates spacers efficiently in vitro18; other Cas proteins (such as Cas9 and Csn2) have accessory roles in the biogenesis phase of prespacers17, 23. Here we present four structural snapshots from the type II–A system24 of Enterococcus faecalis Cas1 and Cas2 during spacer integration. Enterococcus faecalis Cas1–Cas2 selectively binds to a splayed 30-base-pair prespacer bearing 4-nucleotide 3′ overhangs. Three molecular events take place upon encountering a target: first, the Cas1–Cas2–prespacer complex searches for half-sites stochastically, then it preferentially interacts with the leader-side CRISPR repeat, and finally, it catalyses a nucleophilic attack that connects one strand of the leader-proximal repeat to the prespacer 3′ overhang. Recognition of the spacer half-site requires DNA bending and leads to full integration. We derive a mechanistic framework to explain the stepwise spacer integration process and the leader-proximal preference.
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