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tto icon Constructing a Genome


Introduction

Tremendous. Powerful. A scientific revolution. These are just some of the words that have been used to describe the genome engineering system, CRISPR/Cas9. The tool is so revolutionary, in fact, that discussions of CRISPR and its potential impact have moved beyond traditional scientific outlets like Science and Nature , and into more mainstream ones like Wired and NPR . Although these latter outlets often describe CRISPR as being easier, more efficient and more affordable to use than other genome engineering tools, they typically do not discuss the scientific reasons why.

Given the immense popularity of Addgene CRISPR requests, it is very likely that you have seen the word “CRISPR” float across your desk or e-mails at some point within the last year. Moreover, we understand that not everyone involved in the Addgene process may have a science background. Therefore, in this article we will attempt to introduce, explain, and compare three popular genome engineering tools (Zinc Fingers, TALENs, and CRISPR) by way of analogy, in order to give you a better understanding of this revolutionary new tool.

The Assignment

Imagine you work for the Public Works Department of Greater Metropolis. You have just received information that a popular travel show, Roads of Our Nation (“RooN”), would like to feature the famously pristine streets and homes of Greater Metropolis and that they would like to begin filming soon. Unbeknownst to RooN, you happen to know there is a portion of a certain road that is rather unbecoming with too many potholes and unsightly images permanently drawn in the cement. Since a RooN feature could be a huge boon for the area, you want to ensure that Greater Metropolis is depicted in its best light. Your assignment is to hire a construction crew(s) to destroy and repave the offending portion (“OP”) before RooN arrives.

The Location of the OP

Unfortunately, one of the quirky “charms” of Greater Metropolis is that none of the roads or streets have names (at least not officially), so you are unable to provide the OP’s location via street address or cross streets. Your best bet is to describe its location by using the abutting houses and fences. Nonetheless, this also presents its own complications. For better or worse, all houses in Greater Metropolis are painted only one of four colors (Green, Tan, Cyan and Amber). Moreover, in order to adhere to Greater Metropolis’ official color motif, Green houses must sit opposite across the street of Cyan houses; and Tan houses must be opposite of Amber houses. Finally, thanks to the fence fad of ‘61, every three adjacent houses are fenced in together, creating a patchwork of different types and colors of fences (more on this later). The picture below illustrates the OP’s location in relation to the abutting houses:

house-example-1


Legend (Analogy/Biological Referent)

  • OP - offending portion / location of DNA double stranded break
  • G - green house / guanine nucleobase
  • C - cyan house / cytosine nucleobase
  • T - tan house / tyrosine nucleobase
  • A - amber house / adenine nucleobase
  • xxxx - Chain link fence / codon
  • === - Wooden rail fence / codon
  • vvvv - Vinyl picket fence / codon
  • ‡‡‡‡ - Iron bars fence / codon
  • - fence posts

Choosing a (De)Construction Company

In the past, the Public Works Department has contracted with the following companies:

  1. ZFN, Ltd.
  2. TALENted Construction
  3. CRISPR Construction

While all three companies can get the job done, each has its own methods, efficiencies, and costs.

ZFN, Ltd. 1

Company Information

ZFN is the oldest of the three companies and has amassed a fleet of vehicles over the years. While this is partially due to the company’s longevity, it is mostly due to the quirkiness of Greater Metropolis and its residents. Shortly after the fence fad subsided, ZFN realized the types and colors of each fence surrounding each set of houses adhered to a common set of principles. The rightmost house of the set (when facing the street) always determined the type of fence (vinyl picket, wooden rail, chain links, or iron bars); the center house chose its color as the color of the pickets / rails / links / bars; and the leftmost house chose its color as the color of the fence posts. 2 Accordingly, while there are well over 500,000 houses in Greater Metropolis, there are only sixty-four different types of fences. To take advantage of this, ZFN has developed sixty-four different devices, each specifically attuned to the properties of one of the sixty-four fence types. Because these devices must scan the entire Greater Metropolis area and its multitude of fences, each device is fairly sizable and must be transported on a wheeled platform bed the length of three houses.

Construction Methods

Given that any one of the sixty-four types of fences could appear multiple times throughout the Greater Metropolitan area, ZFN requires the Public Works Department to describe the fences in a specific order to narrow down the intended construction site. See the completed prompts below:

fences-explanation-list


Once ZFN has this information, it locates and assembles two construction crews (one for each side of the street). Each crew drives its own construction vehicle, which pulls several platform beds carrying the applicable fence-locating device in the order described above. By using a list of ordered fences for both sides of the street and the applicable devices, ZFN is usually able to locate the intended construction site. In an area as large as Greater Metropolis, there is always the chance that this exact combination may appear elsewhere. If there is such a concern, the Public Works Department can list additional fences. However, this could increase the likelihood of some of the potential drawbacks described in the next section. Assuming that both construction crews are able to locate, reach, and park at their respective locations, construction will begin once the two vehicles have been tethered to one another, in order to secure the drilling equipment. The picture below illustrates a typical ZFN construction site:

house example 2

Pictured above are two construction crews, each pulling three platforms of fence-detecting devices. While the construction vehicles are depicted between the houses for this analogy, in many scientific renderings, zinc finger nucleases bind to the external side of DNA, or, in this case, behind the houses. 3

Drawbacks

ZFN is not always the most timely or affordable company with which to work. It can take days for ZFN to locate the appropriate platform beds and devices in its garages and lots. Sometimes, the appropriate device is unavailable or is no longer optimized for the Greater Metropolis area. In either case, the applicable device must be specially-ordered and/or manufactured, which, if time is of the essence, can add time and costly expenses. Even when all platform beds and devices are available, ZFN must still have them brought to their main facility for construction vehicle-line assembly to ensure that each line has the appropriate beds attached for locating the specific order of fences. Moreover, if there is a parked car or some other obstruction along one of the fences, not only will the vehicle line be unable to park but it may not even be able to locate the proper fence. The picture below illustrates this issue:

house example 3
In the picture above, a car (circled in red) is parked in front of one of the fences. The parked car impairs the crew’s ability to not only park at the site but also locate it.


Finally, even if one crew lines manages to find the OP and park, construction cannot begin until both crews arrive.

TALENted Construction 4

Company Information

TALENted has been around for several years now. In that time, they, too, have figured out a way of dealing with the quirks of Greater Metropolis. Rather than using ZFN’s sixty-four separate fence-locating devices, TALENted has created four devices that are able to identify and locate houses based on their color. While the devices can be transported on a shorter platform bed (length of one house), they are about three times taller than the fence-locating devices and require additional rigging to keep in place. These new devices have, however, provided TALENted with several advantages. For example, instead of having to manufacture and maintain hundreds of different devices and vehicles to account for each of the sixty-four fences with a few backup devices, TALENted can amass hundreds of devices and vehicles with several backup devices. This cuts down not only on manufacturing, development and personnel training costs, but it also provides TALENted greater flexibility in the types and numbers of jobs it can take.

Construction Methods

Given that there are only four colors used in all of Greater Metropolis--green, tan, amber and cyan, TALENted requires the Public Works Department to describe the houses in a specific order to narrow down the intended construction site. See the completed prompts below:

House construction list

TALENted’s construction methods are similar to ZFN’s. Once TALENted has the information above, it locates and assembles two crews (one for each side of the street). Each crew drives its own construction vehicle, which pulls several platform beds carrying the applicable house -locating device in the order described above. The list of eighteen houses usually allows TALENted to locate the intended construction site. However, as with the fences, in an area as large as Greater Metropolis, there is always the chance that this exact combination of houses may appear elsewhere. While ZFN’s and TALENted’s location methods may differ, they both use the same drilling equipment. Accordingly, assuming that both construction vehicles are able to locate, reach and park at their respective locations, construction will begin once the two vehicles have been tethered to one another, in order to secure the drilling equipment. The picture below illustrates a typical TALENted construction site:

talen construction site example

NOTE: While the construction vehicles are depicted between the houses for this analogy, in many scientific renderings,TAL effector nucleases bind to the external side of DNA, or, in this case, behind the houses. 5

Advantages and Drawbacks

In the past, TALENted has been a slightly easier and more affordable company than ZFN with which to work. Because of the redundancies within their fleet, delays in obtaining the correct device and platform are rare. While assembly of the respective lines must still occur at TALENted’s facility to ensure that the devices and platform beds are properly ordered, the fact that there are only four types of devices and platforms generally makes this easier and quicker.

There are, however, still some concerns. Given the fact that TALENted’s devices are nearly three times taller than ZFN’s, navigating Greater Metropolis’s narrow streets and tunnels can make getting to the intended construction site much more difficult.

Additionally, although TALENted requires fewer types of devices than ZFN, a greater number of TALENted’s devices are needed. This can be both helpful and harmful at the same time. For example, if one of the eighteen houses on the list is being fumigated and cannot be located due to the fumigation tent, the remaining seventeen devices may still be able to successfully locate the intended construction site. The picture below illustrates this:

talen construction site example


On the other hand, given the number of redundancies within TALENted’s fleet, some malfunctioning devices may go unnoticed. It is not rare for one device, in a line of nine, to malfunction en route to the site. The device may begin tracking the wrong house or cease functioning altogether. In some cases, the driver may even purposely ignore the device and choose the most suitable spot. This can cause the construction line to arrive at an incorrect location. See the picture below:

talen construction site example of incorrect arrival


However, the scenario above is less likely if more houses are provided, which reduces the chances of the house order repeating elsewhere.

CRISPR Construction 6

Company Information

CRISPR Construction is a much younger company than both ZFN and TALENted. Nevertheless, CRISPR has figured out how best to utilize its limited resources in Greater Metropolis. Rather than expending substantial funds on the manufacture, maintenance and storage of hundreds of devices and platforms, CRISPR has invested heavily into training its employees and photo-documenting the Greater Metropolis area. Rather than narrowing down a site’s location based on fences and house colors, CRISPR can use a submitted panoramic snapshot (containing a sufficient number of houses in the backdrop) as a guide to trace the proposed site to its location.

Construction Methods

Unlike ZFN and TALENted, CRISPR does not need to laboriously contact and assemble two construction crews with vehicle lines the length of a small neighborhood. The company can almost immediately send out a single, specialized construction vehicle upon receiving the requisite construction site snapshot. Using the snapshot as a guide, the driver can track the OP to its location. However, because CRISPR makes uses of specialized construction equipment, there must always be a certified Project Area Manager (PAM) near the intended site before construction can begin. The picture below illustrates a typical CRISPR construction site:

crispr construction image
In the picture above, a single construction vehicle is needed at the OP. Note, however, that the PAM (picture to the right of the vehicle atop a platform) must be onsite for construction to begin. While the construction vehicle is depicted between the houses for this analogy, in many scientific renderings, the CRISPR Cas9 system surrounds the DNA, or, in this case, surrounds the houses. 7


Additionally, CRISPR has a variety of specialized construction vehicles to choose from, depending on the nature of the job. However, in each case, there must be an appropriately certified PAM to supervise the construction.

Advantages and Drawbacks

Given the nature of CRISPR’s location methods, the advantages in time and costs saved can be immediately appreciated. There is no need for creating long descriptive lists; manufacturing or obtaining specific tracking devices; and/or assembling long lines of platforms. Moreover, because CRISPR does not need to worry about parking said platforms, CRISPR can operate on much shorter streets than ZFN and TALENted. In some cases, depending on the street and the type of construction vehicle and PAM, CRISPR can even operate at multiple sites along the same street.

Nevertheless, CRISPR does have a few drawbacks. The construction vehicles used are much larger than the individual construction vehicles of ZFN and TALENted, so getting to the site can be a little more challenging. However, because ZFN and TALENted require two construction vehicles onsite, CRISPR’s vehicle is effectively smaller or equal to the other companies onsite. Another concern is offsite construction. Like ZFN and TALENted, there is a possibility that CRISPR will operate at the wrong location. Depending on the snapshot’s quality, the CRISPR driver could mistake (or ignore) one of the houses in the snapshot and wind up at the wrong location. However, this is mitigated by the fact that there must be a PAM nearby for construction to begin.

Assessment

CRISPR provides an affordable and efficient option in dealing with the OP. Given the short timeline that the Public Works Department has to work before RooN arrives, CRISPR likely provides the best option in this case; provided, however, that the appropriate construction vehicle can be identified and the applicable certified PAM is available. Fortunately, the job market has exploded recently, providing CRISPR with more available PAMs for additional construction sites, so it is likely that CRISPR will be able to fulfill the Department’s needs in a timely manner.

Repaving the OP 8

Regardless of which construction company the Department chooses, it is vital that the road be immediately repaved under proper direction and supervision. A team of road pavement specialists has provided the Public Works Department with a template for ideal road conditions. This template must be given to and executed by the chosen construction company to ensure optimal filming by the RooN crew. Failure to do so could lead to some unwanted consequences for the RooN taping. A few such consequences are discussed below.

  • Detours. If the site of the OP is not properly paved, RooN may wind up having to take a detour. This would mean that several of the nicer homes on that street fail to make the film reel.
  • Unflattering Shots. The Public Works Department wants RooN’s depiction of Greater Metropolis to be that of a smooth, comfortable ride through the city. This idyllic depiction could be easily spoiled by severe camera lurches and tumbling if the RooN crew chooses to drive through an improperly paved and bumpy site.
  • Liability. Moreover, should the RooN crew choose to drive through an improperly paved site, there is a risk that the camera van may not even make it through the site. Punctured tires and damage to the van’s frame could easily bring filming to a halt. Not only would Greater Metropolis lose out on some good PR but it might end up with a lawsuit.
  • Resident Remedies. If the site goes unpaved for too long, the residents of Greater Metropolis have been known to use any available resources to address these issues. For example, unsightly wood planks have been used to create makeshift bridges between paved portions. This is not the image that the Public Works Department wishes to convey.
  • Risks to Concrete. Without proper oversight during the repavement process, there is always the risk that crude and other unwanted images may be drawn in the drying concrete. This is also something the Public Works Department would like to avoid.
  • Unwanted Features. Additionally, without proper direction and planning, the repaved OP site may unintentionally wind up with a speed bump or crosswalk. While a crosswalk would not affect the actual filming of the area--the van passing over the painted walk silently and the RooN crew unlikely to make any mention of it, a speed bump would affect how the crew films its shots and how residents drive through the area.

Given the strong interest of the Greater Metropolis administration in presenting a picturesque setting for the RooN crew and future audience, the Public Works Department needs to ensure that whatever company is hired that the OP is repaved pursuant to the template under proper direction and supervision.

Summary

Hopefully this analogy has helped you gain a better understanding of what advantages and benefits the CRISPR / Cas 9 genome engineering system offers. For example, providing a snapshot of the construction site (protospacer information) to the construction company costs less time and money than developing or obtaining specific devices (zinc fingers or TAL effectors) for fences (codons) and house colors (nucleotides), respectively. Scientists that either lacked the expertise or funds to develop or obtain these tools now have readily available access to an efficient genome engineering tool. Moreover, instead of having to reassemble two new construction crews and their respective platforms and vehicles (zinc fingers and TAL effectors with associated endonucleases) for every new construction site, the CRISPR company simply uses the panoramic snapshot (tracr RNA) to guide it to the intended site.

Every day, scientists continue to research and develop new ways to utilize the CRISPR / Cas 9 system. From actively treating genetic diseases, such as leber congenital amaurosis which can cause blindness 9 , or combating new viruses like Zika 10 , to developing more drought-resistant crops 11 , CRISPR has had a significant impact across a variety of scientific fields. With so many promising results already, we, too, look forward to seeing what future applications CRISPR will provide further down the road.

Footnotes

  1. For the technical explanation of ZInc Finger Nucleases, please see The Zinc Finger Consortium, “Scientific Background,” http://www.zincfingers.org/scientific-background.htm (last visited July 1, 2016).
  2. table of fence iterations
    While specifying every possible fence iteration is not necessary for the purpose of this discussion, we have provided the following key for the curious reader.
  3. figure of zinc finger system binding to DNA
    The illustration above represents how zinc fingers bind to DNA.
  4. For the technical explanation of the TALEN technology, please see Addgene, “Addgene’s Guide to TALEN Technologies,” http://www.addgene.org/talen/guide/ (last visited July 1, 2016).
  5. figure of TALEN system binding to DNA
    The illustration above represents how TALENs bind to DNA.
  6. figure of crispr system binding to DNA
    The illustration above represents how CRISPR/Cas9 binds to DNA.
  7. For the technical explanation of the CRISPR/Cas9 system, please see Addgene, “CRISPR/Cas 9 Guide,” http://www.addgene.org/crispr/guide/ (last visited July 1, 2016).
  8. This repaving analogy refers to Homology Directed Repair, see Addgene, CRISPR 101 Homology Directed Repair, http://blog.addgene.org/crispr-101-homology-directed-repair (last visited July 1, 2016), and Non-Homoglous End Joining, see Addgene, CRISPR 101: Non-Homologous End Joining, http://blog.addgene.org/crispr-101-non-homologous-end-joining (last visited July 1, 2016).
  9. Regalado, Antonio. CRISPR Gene Editing to be Tested on People by 2017. MIT Technology Review (Nov. 5, 2015), available at https://www.technologyreview.com/s/543181/crispr-gene-editing-to-be-tested-on-people-by-2017-says-editas.
  10. Flam, Raye. Fighting Zika Virus with Genetic Engineering. Bloomberg (Feb. 4, 2016), available at http://www.bloomberg.com/view/articles/2016-02-04/fighting-zika-virus-with-genetic-engineering.
  11. Tuberosa, R. & Salvi, S. Genomics-Based Approaches to Improve Drought Tolerance of Crops. Cell 11(8), pp. 405-412.