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This model illustrates the role that CRISPR plays in defending bacteria from infection by focusing on a single cell surrounded by an unseen environment. In natural environments bacteria are challenged with infectious viruses very often, so bacteria must have ways to defend themselves in order to survive.
There are many ways in which bacteria defend themselves from viruses. Bacterial defense systems can be broadly categorized as innate and adaptive. Innate systems are generally exploit a feature common to many or all viruses, and do not change based on the bacterium's past. A common example of this would be restriction enzymes, which slice up nucleic acids containing certain base sequences, regardless of where the nucleic acid is from. This is also analogous to our human innate immune system, where white blood cells like macrophages and neutrophils attack foreign material using nonspecific methods like reactive chemicals.
Bacteria were recently found to also employ adaptive immunity in their constant struggle against viruses. This adaptive system is called CRISPR. With it, bacteria are able keep a heritable genetic record of past infection attempts, which makes them and their descendants much more effective at fending off future infections by the same or similar viruses. This is made possible through a system that specifically recognizes the DNA of past viruses using DNA samples called spacers stored in a CRISPR array and cleaves it using a targeted protein complex.
CRISPR is often very effective in protecting bacteria from infection. However, there are many components to the system that must work together to provide this protection. All of the components are coded for in a single locus, often found on a plasmid separated from the main genome. The structure of this locus is shown below: The whole locus is transcribed together, moving from left to right in this diagram. After transcription, the Cas genes are translated into various Cas proteins, and the spacers in the CRISPR array are processed into guide RNAs.
The spacers in the CRISPR array are short fragments of DNA from viruses that the bacterium or its ancestors encountered in the past. When they are processed into guide RNAs, they are critical for the function of one of the protein components, Cas9. Cas9 proteins bind to one of these guides and uses it to recognize when other DNA might come from the same virus as the original spacer in the array. If the guide and the foreign DNA sequences match, indicating it is probably also a virus, the Cas9 protein is able to cut the DNA and destroy it.
Other Cas proteins are important for maintaining the library of spacers in the CRISPR array. Four Cas1 monomers and two Cas2 monomers can form a protein complex which is capable of taking small sections of DNA from viruses and adding them to the CRISPR array as new spacers. When this happens, the new spacers are added at the 'front' of the array, just after the leader sequence (see the diagram above). This means that the array is roughly ordered so that the most recently encountered viruses are closer to the Cas genes in the locus and are transcribed first. Spacers can also be lost from the array. This tends to be more likely the closer the spacer is to the end of the array, meaning that older spacers tend to be lost first.
The CRISPR system as a whole only works when each of its components is functioning properly, and each component often only works when in conjunction with other components. This model is meant to help illustrate this complex interdependence.
There are several types of dynamic agent in this model: RNA polymerases, viruses, guide RNAs, cas9 proteins, and cas1-cas2 complexes. There are also three types of static agent: the bacterial genome, the CRISPR locus, and the cell wall.
The static agents are created when the model is set up and then serve only as interaction points for the dynamic agents. The cell wall is the ring of gray around the outer edge of the cell, and simply contains the other agents. The genome is the ring of darker blue near the center of the cell, representing the circular genome typical of bacteria. The CRISPR locus is the small band of light blue at the center of the cell, with a slightly lighter promoter region at one end. This represents a plasmid, which is often where CRISPR loci are found. The role of the genome and CRISPR locus will be discussed below.
All of the dynamic agents move through the cell at a speed influenced by their relative size. Small molecules like guide RNAs can diffuse very quickly, moderately sized molecules like proteins diffuse slightly more slowly, and very large molecules like viral DNA move very slowly. Viruses diffuse in the general direction of the bacterial genome, while every other dynamic agent moves via a random walk constrained by the cell wall. All of the dynamic agents also have a small chance to degrade on each tick, which is influenced by a variety of factors.
When RNA polymerases encounter the promoter region of the CRISPR locus, there is a chance that they will transcribe the locus and produce the molecular components of the CRISPR system. They do not typically have perfect processivity, meaning that they may fall off of the DNA before transcribing the entire array. In this case, only the CRISPR components that come before the drop location in the locus will be produced.
During an infection, viruses enter the cell at the cell wall. If a virus reaches the genome, it is able to incorporate into the DNA. At this point the cell is considered lost to the infection.
Cas9 proteins move through the cell until they encounter a free guide RNA. If they are not bound to anything, the Cas9 will bind to the guide RNA and begin to carry it around as it continues to move. There is a small chance each tick for the Cas9 to dissociate from the guide. If the Cas9 encounters a virus while it also has a guide RNA, it tries to cut the virus. If the guide matches the virus, the virus will be destroyed, and otherwise the virus is unaffected.
Cas1-Cas2 complexes move through the cell until they encounter a virus. When this happens, they bind to the virus and cut out a sample of it. They then carry the sample to the CRISPR locus in the center of the cell and add a new spacer to the array that matches the invading virus.
This model can be used to investigate the effects of different CRISPR expression strategies. These strategies can involve changes to the overall expression of the locus or changes to the persistent expression of spacers from less recent infection attempts.
This model can also examine how the efficiency of CRISPR is affected by the features of the environment or the invading virus. For example, it is easy to compare how effective CRISPR is at stopping very recently encountered viruses or viruses that are more rare. Similarly, it is possible to see how effective CRISPR becomes when there is an increase or decrease in viral diversity or a change to the array size.
Initializes variables and creates the cell and the CRISPR system's components.
Runs the model.
Infects the cell with a random virus.
Infects the cell with a common or recently seen virus, as determined by the cell's CRISPR array.
Toggles whether or not guide RNAs and viruses are labeled with their sequence.
Controls the size of the bacterial CRISPR array when the model is initialized.
Controls the diversity of the viruses that the cell encounters.
Controls the processivity of the cell's RNA polymerase. The value of the slider is the probability that the polymerase will fall off of the DNA before finishing transcription of the next spacer.
Controls the level of expression of CRISPR in the cell. The value of the slider is the probability that an RNA polymerase will transcribe the CRISPR locus when it encounters its promoter.
Plots the number of each component of the CRISPR system in the cell over time. These components include guide RNAs, Cas9 proteins, and Cas1-Cas2 complexes. The number of Cas9 proteins currently bound to a guide RNA is also plotted.
GUIDE RNA COUNT
Displays the current number of guide RNAs in the cell.
Displays the current number of Cas1-Cas2 complexes in the cell.
Displays the current number of Cas9 proteins in the cell.
Displays the current number of viruses in the cell.
Plots a histogram comparing the frequency of each guide RNA type currently in the cell (black bars) and the frequency of each viral spacer in the CRISPR array (red bars).
BACTERIAL CRISPR ARRAY
Shows the current state of the bacterium's CRISPR array. Viral spacers at the left or front of the list are the most recently added.
Displays the current length of the bacterium's CRISPR array.
When the model is initially set up, all of the guide RNAs and Cas9 proteins are unbound. After starting the model, Cas9s quickly become bound to guide RNAs. Does every Cas9 have a guide RNA? Why is this the case?
Look at the CRISPR Elements plot or the monitors below it and compare the number of guide RNAs to the number of Cas9 proteins. Why is it advantageous for the cell to have more of one than the other?
Look at the CRISPR Elements plot or the monitors below it and compare the number of Cas9 proteins to the number of Cas1-Cas2 complexes. Why are there fewer Cas1-Cas2 complexes, even though each gene in the locus is transcribed the same number of times?
Look at the Guide RNA histogram. The red bars show the frequency of each type of spacer in the CRISPR array, and the black bars show the frequency of the resulting guide RNAs in the cell. If two types of spacer each occur in the array the same number of times, do they always result in the same number of guide RNAs being produced? Similarly, if one type of spacer occurs in the array many times, does it ever result in fewer guide RNAs than a spacer type that only occurs a few times? In each of these cases, what makes some spacers result in more guide RNAs than others? It may help to also look at the Bacterial CRISPR Array monitor below the plot.
RANDOM-INFECTION buttons allow the user to test how the cell fights off different types of viruses. The
RANDOM-INFECTION button introduces a random virus within the level of diversity specified by the
MAX-VIRUS-TYPES slider into the cell. The
COMMON-INFECTION button introduces a virus that the cell has seen either very recently or very often (suggesting it is common in the environment) into the cell. Is there a difference in how often these two types of infection succeed? Why do you think that is?
How does changing the amount of viral diversity in the external environment influence the cell's ability to fight off infections? This can be accomplished using the
MAX-VIRUS-TYPES slider. Does it make a difference for both random and common infections, or just one of these infection types?
Changes to the cell's regulation of CRISPR can have a large influence on its efficacy. This can be seen by manipulating the
CRISPR-FUNCTION sliders. Try to understand what effect each slider has individually. First, the
GUIDE-UNBIND-CHANCE slider sets how likely it is for guide RNAs to unbind from Cas9 proteins on each tick. Why do you think this might make a difference to the cell? How does changing this slider affect the proportion of Cas9s bound to a guide RNA? Secondly, the
PROCESSIVITY slider changes how likely it is for RNA polymerases to continue on after finishing transcription of a particular spacer in the array. How does changing this slider affect the number of guide RNAs in the cell? On the Guide RNAs histogram, can you tell why this number changes? Which spacers are most strongly affected by it? Finally, how do the levels of the CRISPR components in the cell change when you increase or decrease the
After investigating how using these sliders affects the state of the cell, test how these changes affect the effectiveness of the CRISPR defense system. Do any of these changes affect all types of infections equally? Do some have a greater effect on common infections than random infections or vice versa?
One simple extension to the model involves the way viruses are treated after being inactivated. If an invading virus is cut up either by CRISPR or by an innate defense system, it takes some time for the resulting inactive fragments to be completely disassembled. Cas1-Cas2 complexes are able to add spacers during this period, increasing the likelihood that a virus could be fought off in the future. However, the current model treats these fragments as being fully disassembled almost instantly.
This model currently focuses on the role of CRISPR in bacterial defense, and only treats the innate defenses abstractly. An updated version of the model could explicitly include different innate bacterial systems. It could also more accurately treat the cell's ability to direct CRISPR components to act non-randomly, for example by recruiting Cas9 or Cas1-Cas2 complexes to the site of an infection.
Similarly, in this model, viruses are mostly passive invaders, simply diffusing inwards until they reach the genome. In reality, they can be transcribed earlier that that, and this can allow them to actively fight back against bacterial defenses. One interesting extension of this model would be to include more active viruses to showcase the interplay between bacterial defenses and viral evasion tactics.
The model also focuses on the initial stages of a viral infection, but CRISPR can continue to have interesting effects in the later stages. For example, even if the bacterium succumbs to the infection, CRISPR may be able to destroy newly produced viruses before they can be released. To see these effects, the model could be expanded to a longer infection timeline, including viral inhibition of normal transcription, production of new viruses, and using a different criterion for determining the end of an infection.
Another interesting effect that follows naturally from a longer infection timeline would be a treatment of lysogenic infections. Currently, all viruses are assumed to be lytic, meaning when they infect a cell they reproduce until the cell dies and releases the new viral particles. While this is true for many viruses, there are also some which are lysogenic. These viruses can insert their DNA into the bacterium's genome. This allows them to 'reproduce' when the bacterium divides, and then revert back to the lytic process described above when their host is in poor health. After the viral DNA is inserted into the bacterial genome, there are several strategies the cell can employ based on its different regulatory machinery. For example, CRISPR may cleave the cell's genome, resulting in the bacterium's death, or simply suppress viral replication when the virus attempts to become lytic again. Modeling these regulatory 'decisions' could be very interesting.
This model has specific turtle shapes that are able to combine with the shapes of other turtles to form new shapes when they are interacting. For example, when proteins bind to nucleic acids (guide RNAs or viruses), their shapes interact so that the proteins appear to be carrying the bound nucleic acid. This functionality relies on using the agent breed declarations to specify the z-ordering of how they are displayed. Specifically, agent breeds that are declared first (closer to the top of the file) will always be displayed 'under' breeds that are declared later (closer to the end of the file).
This model employs a supplementary function to improve auto scaling behavior for histograms. By default, histograms only autoscale the y-axis. The
update-histogram-ranges function used here implements x-axis auto scaling which can increase but never decrease the range, similar to the default y-axis auto scaling. It also implements y-axis scaling which can increase or decrease the plot's range. This makes the histogram always take up most of the plot area while keeping the entire histogram visible and minimizing the number of rescaling events.
CRISPR Ecosystem CRISPR Ecosystem LevelSpace CRISPR Bacterium LevelSpace
The image of a CRISPR locus was adapted by Merry Youle of the Small Things Considered blog for their article Six Questions About CRISPRs from Sorek, R., Kunin, V. & Hugenholz, P. CRISPR - a widespread system that provides acquired resistance against phages in bacteria and archaea. Nature Reviews Microbiology 6, 181-186 (2008).
For general information about CRISPR, visit https://sites.tufts.edu/crispr/ or see the review below:
For information relevant to possible extensions to the model, see the articles below:
If you mention this model or the NetLogo software in a publication, we ask that you include the citations below.
For the model itself:
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Copyright 2019 Uri Wilensky.
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