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CRISPR: Accelerating the pace of molecular biology

CRISPR stands for Clusters of Regularly Interspaced Short Palindromic Repeats. Dr. Jennifer Doudna was one of the first researchers to see these short palindromic repeats in bacteria and archaea where she speculated that they were being used as a form of molecular immune system to protect these organisms from viruses.

Even bacteria get sick, so having a protection against invading viruses is a matter of life and death to a cell. Recall that viruses are essentially genetic material that will reproduce itself again and again after it hijacks a cell. Viruses may have protein coats or membranes to protect them outside of the cell, but inside, they are little more than DNA. If this DNA can be damaged or destroyed, then the virus is rendered harmless.

Screen Shot 2015-07-27 at 9.54.10 PMTo the right is a clip from Dr. Doudna’s video illustrating the repeated elements (in black) flanking a variety of ‘other DNA’. This ‘other DNA’ is what the cell will use to identify  foreign DNA – presumably from retaining the genomic material from an earlier exposure either in the lifetime of the single cell or its parents.

So, how does it actually work?
Two videos do an excellent job of explaining how CRISPR works. A short, simple video from MIT gives a non-technical explanation (a good place to start).

MIT’s McGovern Institute

Jennifer Doudna explains the system in greater detail…

Basically, the natural system uses two RNA molecules to target specific DNA sequences in the genome and recruit a protein that acts as an endonuclease to cleave this target:

crRNA – a ‘targeting’ molecule
tracrRNA – an adaptor RNA that recruits CAS9 to the bound crRNA
CAS9 – an endonuclease enzyme that will bind and cleave DNA once recruited by the RNAs

Doudna’s lab improved the system by combining the two RNA molecules into a single RNA that still effectively recruits CAS9 but is easier for researchers to manipulate in the lab. This last element is essential because manipulating this RNA sequence gives researchers the power to target any DNA sequence in the cell.

As stated above, the system was originally identified in prokaryotic organisms where it appears to allow targeting of the viruses that attack them. CRISPR uses ‘stored’ DNA as the targeting RNA and then brings in CAS9. CAS9 binds to the targeted DNA and cleaves it resulting in one of two possibly outcomes. 1) the virus is destroyed and is no longer a problem, 2) the virus is cut, but then repairs itself – hopefully in a way that introduced fatal mutations.

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How might this translate into clinical medicine?
The possibilities are endless, however a few low-hanging fruit present themselves immediately. Among these are therapies for sickle cell anemia (and a host of other blood disorders). Because sickle cell anemia is caused by a single base pair mutation, it is conceivable that hematopoietic (i.e. blood) stem cells can be isolated, the faulty gene repaired, and then re-introduce the corrected stem cell back into the body (possibly after the faulty stem cells have been ablated).

The newly altered and re-introduced stem cells now do the rest of the work for you by finding their place in the body where they reside while continually producing cells with the desired genetic changes.

The key is that these RNA molecules are quite simple to make exactly and in pure form (i.e. they can be manufactured chemically rather than needing cells to do the job for us and then we have to clean up all the extraneous contaminants). Most labs will design the molecules in-house and then order the constructed molecules from a ‘core lab’ that specializes in doing just that.

Jacob Corn, of UC Berkeley has compiled a simple protocol that anyone with a modicum of molecular biology training could follow. Find that protocol here.

 
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Posted by on July 27, 2015 in Uncategorized

 

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More on the Lac Operon

A while ago I wrote two posts about the Lac Operon here. The first pointed to an animation by McGraw Hill Publishers that did a pretty good job illustrating how the operon works. In the second post, I highlighted the notion of polycistronic messages (more than one gene per mRNA molecule) and how this allows for control of a number of related genes at once – a trait not shared by eukaryotic cells. In that second post, I also finished with a graph of how cells grow in the presence of glucose and lactose.

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Cell Growth in the presence of glucose + lactose – As glucose is depleted, cells adjust to lactose digestion

One feature of that graph (reproduced here) that is notable is a little bump in the growth rate as glucose runs out and the cell converts to lactose digestion. A second important feature is that the rate of growth slows when the cell is burning lactose as its primary fuel.

 

Together, these features suggest that the cell is regulating lactose digestion very closely. In fact, there are two primary mechanisms of this regulation to appreciate. The first is that the lactose-digesting enzymes are controlled together on an operon that is regulated by lactose itself (or at least we can assume so for simplicity’s sake). In the absence of lactose, no lactase enzymes are made and no lactose is used as fuel. The reason for this is obvious when you look at the slope of cell growth under glucose metabolism (left) and lactose metabolism (right). Clearly, growth is SLOWER when lactose is used as fuel.

Therefore, so long as there is glucose, it is pointless to digest lactose at the same time. So it is best to only turn on the lac operon in the ABSENCE of glucose – regardless of whether lactose is present of not.

If glucose is absent and lactose is absent, turning on lactase enzymes is still useless. However, slow growth is better than no growth. So we should have a mechanism to turn on the operon when there is lactose in the environment.

Here’s a matrix of ideal regulation:

Screen Shot 2014-03-31 at 2.18.05 PM

How can a little, mindless bacteria achieve this exquisite control?

Simple: By using two regulators. One for glucose and one for lactose. Only when both conditions (glucose-, lactose+) are met do we make lactase.

Structure of the Lac Operon

operon1

First, lactose itself serves as an inducer. In the absence of lactose, a regulator protein binds to a DNA site between the polymerase binding site (the promoter) and the structural genes (the enzymes). When the regulator binds, its presence physically prevents the progress of RNA Polymerase.

When lactose is present, it binds the repressor protein in a way that causes its shape to change in a way that can no longer bind the DNA. The repressor then drifts away from its binding site allowing RNA Polymerase a clear shot to the structural genes.

operon2

However, RNA Polymerase is not always parked on the promoter waiting for the repressor to be removed. Its binding requires another protein to help stabilize its interaction with the DNA. This second protein is the CAP protein. The Catabolite Activated Protein. However, CAP alone will not bind either. It requires a signaling molecule called cyclic AMP (cAMP). cAMP is readily broken down when glucose is in the cell, so it only accumulates when glucose is absent. In that case, cAMP accumulates and binds to the CAP protein, which then binds to the CAP site. This site is located adjacent to the promoter, but on the side away from the structural genes. When CAP binds, it assists in recruiting the RNA polymerase to the promoter.

operon3

Therefore, if only one condition is met, it is insufficient to promote gene transcription. Only when the CAP+ cAMP protein is bound will the Polymerase be recruited. And only when lactose is present, will the repressor protein let the Polymerase pass.

operon4

In terms of the matrix we set forth above, we can see that these molecular interactions result in exactly the regulation that is optimal:

post-operon

 

 
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Posted by on March 31, 2014 in Uncategorized

 

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A quick followup on that lac operon post

Last week I posted a quick link about operons for my micro class to check out before taking their quiz on bacterial gene regulation This post is intended to complement that one. To go back to that post, click here. If there’s one thing to remember about operons it is that bacteria, lacking a nuclear membrane, regulate their genes differently than Eukaryotes. Having a nuclear membrane separates transcription and translation into two distinct compartments allowing for more subtle tweaking of Eukaryotic mRNAs before they are exported for translation.

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Click on this figure to go to a good description of how polycistronic genes work

One thing this does is it makes it very beneficial to package genes with related function closely on the genome and use a single regulatory region to control them all together. They wind up getting packed so closely together that they are actually expressed as a single messenger RNA – known as a polycistronic (meaning ‘many gene’) message.

Upstream of this polycistronic cassette are regulatory elements. One element common to all regulatory elements is the promoter. The promoter consists of several elements which ‘promote’ the binding of an RNA polymerase to the DNA. Additional regulatory elements exist to ensure that this polymerase only transcribes the genes if they are needed. In doing so, the cell conserves energy and components (e.g. Amino Acids) for only necessary processes.

In the case of the paradigm lac operon, lactose is a fuel source, but not as good as glucose. Therefore, enzymes to digest lactose are only needed when lactose is present, but glucose is not. In order to interrogate both conditions, two additional regulatory elements are present.

First, the operator sequence. This sequence binds a repressor protein that physically blocks the polymerase’s path in the absence of lactose. However, if lactose is present, the sugar binds to the repressor, causing a conformational (shape) change that causes the protein to release its grip on the operator sequence.

Second, a catabolite activator protein (CAP) will only bind to the DNA behind the RNA polymerase if cAMP is present. Let’s not get too distracted, other than to say that cAMP levels are high in the ABSENCE of glucose, and low when that sugar is present. When cAMP binds to the CAP protein it can now bind the DNA and do it’s other job: making a nice binding site for the RNA polymerase. Without CAP, the polymerase binds very inefficiently.

Together, the production of lactase enzymes (those that digest lactose) is exquisitely controlled in a way that conserves the most energy.

ImagePs – take a look at this graph and tell me why (not mechanistically, but rationally) the cell does not make lactase enzymes when both glucose and lactose are present.

 
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Posted by on March 15, 2014 in Uncategorized

 

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Lac Operon

I’m typically not a fan of McGraw-Hill’s animations because they are rather dry and un-engaging, however, this one does a good job of describing conditions under which the lac operon will be either ‘on’ or ‘off’ and illustrating how positive and negative regulatory elements work together so that the operon is on when needed, but conserves energy by remaining off when it is not needed.

http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter12/animation_quiz_4.html

more on this later….

 
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Posted by on March 10, 2014 in Uncategorized

 

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A damn fine cup of coffee

I was looking through my next lecture for Microbiology (which I realize I still need to post online) and was reminded of an older post that I made pointing to a virtual lab on streak plating of bacteria.

I recommend my students check that out if they have not plated bacterial cultures in the past. You can find that post here.

Also, as always this time of year, I like to reflect on some of the great pleasures of life. And, because these things often become conversations in my classes, I’d like to share a short video that couples two of my favorite things: Coffee and Twin Peaks

 
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Posted by on January 15, 2014 in Uncategorized

 

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A thoughtful article on the use and abuse of antibiotics

ImageEarlier today, I found this well written article on the era of antibiotics by Maryn McKenna (Published November 20, 2013). While I’m not sure I agree with everything in it – and have been spending time tracking down some publications to support or refute some data cited here (particularly in regards to the use of antibiotics in agricultural animals), the  summary of how antibiotics were first discovered and used and how researchers including Flemming feared an end of antibiotic usefulness, paints a vivid portrait of the problem at hand.

While we might typically think of antibiotics as being prescribed in a clinic following a positive test for strep throat or some other bacterial infection, that is just one example of their use. One element of this paper that I found particularly insightful was how easily overlooked are the myriad uses of antibiotics in situations such as surgical procedures or following chemo- / radiation therapy.

 British health economists … recently calculated the costs of antibiotic resistance. To examine how it would affect surgery, they picked hip replacements, a common procedure in once-athletic Baby Boomers. They estimated that without antibiotics, one out of every six recipients of new hip joints would die.

Let me know what you think.

 
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Posted by on November 24, 2013 in Uncategorized

 

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Paul Offit’s Vaccine class

ImageIn my microbiology class, students read Paul Offit’s ‘Vaccinated’, an excellent account of the life and work of Maurice Hilleman, creator of many vaccines in common use today. I don’t know how many of my students are aware of it, but Coursera.org is a free online university offering courses in a number of subjects taught by senior faculty from many distinguished universities. Today marked the first day of Paul Offit’s Vaccine course, which covers topics related to the history, development, use and misinformation surrounding vaccines. From the course website:

1) History of Vaccines – Viruses

2) History of Vaccines – Bacteria

3) Current and ‘Alternate’ Schedule

4) Common Questions About Vaccines

5) Vaccines in the media

6) Creation of a Vaccine – Case Study: The Rotavirus Vaccine

7) Vaccine Exemptions

I can’t promise that this course is still open, but I expect it is – and best of all it’s FREE.

Furthermore, if you sign up and only decide to watch one or two class lectures, there’s no risk: just drop out or simply stop signing in. So, if you have any interest in the subject, or would simply like to see what it’s like, go onto Coursera.org and type ‘vaccines’ in the search bar.

 
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Posted by on September 3, 2013 in Uncategorized

 

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