#KSDNA: Genome editing workshop (CRISPRs in Wonderland)

Hello again from wonderful Whistler!

This moose has a Ph.D. in foraging
This moose has a Ph.D. in foraging

I’m having a splendid time at the Keystone Symposia’s DNA Repliaction and Recombination meeting. I’ve heard some amazing talks, and drank MANY teeny-tiny mugs of coffee.

Seriously, Keystone? Scientists require FAR larger doses of caffeine for productive thinking
Seriously, Keystone? Scientists require FAR larger doses of caffeine for productive thinking

I wanted to use this post to give a rundown on a workshop I attended yesterday covering Genome Editing. The workshop consisted of a series of short presentations given by a mixture of junior investigators and researchers from biotech firms. The talks all covered ongoing progress in the field of genome editing. We learned about technologies that sound like they could have been ripped from the pages of a Margaret Atwood novel, yet the reality is that science is stranger and more amazing than fiction.

This novel, specifically. (Which is INCREDIBLE)
This novel, specifically. (Which is INCREDIBLE)
Although the fact that you can buy a CRISPR off the shelf is pretty incredible too.
The fact that you can buy a CRISPR off the shelf is pretty incredible too.

I’m not going to talk about any of the unpublished or proprietary data that I saw at the workshop, but I did want to give a brief summary of the amazing progress we’ve made in custom DNA-tinkering, and the incredible biology behind the innovation. The term “genome editing” could encompass a wide variety of techniques and tools, however, for the most part, the talk focused on current efforts to introduce specific changes into the DNA sequences of living cells. Researchers are already using these technologies to make transgenic organsims (like glowing worms or custom mutant monkeys).

Source: Tzur et al. Genetics, 2013
Source: Tzur et al. Genetics, 2013

The long-term goal for genome editing, however, is to create cures for genetic diseases like sickle-cell anemia or cystic fibrosis. These disorders arise due to very specific mutations in very particular genes. Researchers are trying to figure out a way to alter the offending alleles to healthy sequence inside living cells. This turns out to be an extremely tricky problem. Genomes are enormous: there are three billion base pairs of DNA inside of every human cell. Almost any time you attempt to change one particular region of the DNA you end up messing with something else, somewhere else, which can have disastrous effects.


Genetic engineering gone wrong? As far as I know, the Weekly World News isn't doing biotech research
Genetic engineering gone wrong? As far as I know, the Weekly World News isn’t doing biotech research…

A new technology called CRISPR offers a way to target very specific regions of the genome. The first papers demonstrating genome editing with a CRISPR in human cells were published in Science in 2013. However, I hesitate to call CRISPR a new technology, mostly because humans aren’t NEARLY clever enough to create this amazing tool on their own. Rather, CRISPR is the latest example of Homo sapiens stealing adapting a feature of bacterial physiology that evolved over the course of several billion years.

You think you're SOOOO clever, don't you.
You think you’re SOOOO clever, don’t you.


I should take a giant step backwards to define what CRISPRs are and how they work. These systems are, at their core, a pattern of DNA sequence that is found in the genomes of 40% of all bacteria and 90% of archaea. The region always consists of a few protein-coding genes (named cas for CRISPR-associated) next to an extended series of short repeated sequences, intermingled with an oleo of spacers. These repeats are what gives rise to the awkward acronym CRISPR, which stands for Clustered Regularly Interspersed Short Palindromic Repeats.


They look like this
They look like this

Widespread whole genome sequencing led to the observation that CRISPRs can be found within many bacterial chromosomes, but their function remained mysterious until 2006 when some very clever researchers in the dairy industry realized what this strange segment of DNA does. CRISPRs function as a bacterial immune system to protect microbes from malicious phages.



Phages are viruses that infect bacteria. In the fight against phages, bacteria are hopelessly outnumbered and outgunned. Phages outnumber bacteria by a factor of 10 to 1 and kill up to 50% of the bacterial biomass on earth each day. There are a LOT of bacteria on the planet, but according to the latest estimates there are more phages on Earth than there are stars in the universe. Because bacteria are constantly targeted by phages, they have evolved mechanisms to protect themselves from new invasive elements. CRISPRs are a unique and elegant system that gives bacterial cells inherited memory of encounters with phages, allowing them to efficiently kill off the virus during subsequent encounters. In other words, CRISPRs are a bacterial adaptive immune system.


So how does this chuck of genetic information protect bacteria from phages? After all, a CRISPR is pretty much just a bunch of repeated DNA sequences with interspersed spacers. The magic of CRISPR is that the interspersed spacers match regions within phage genomes. Those spacers in the bacteria’s genome are small chunks of DNA that the bacteria stole from invading phages and (in a process that remains mysterious to this day) stuck onto their own chromosomes. These sequences let bacteria recognize and destroy phage DNA the next time it enters the cell. Bacteria recognize invading phages by first making the whole CRISPR region into one big, long RNA. That RNA gets cut up into individual pieces by the CRISPR-associated proteins. Each of the little bits of RNA binds to a collection of proteins, which then go into seek-and-destroy mode. If the proteins and RNA bind to a matching piece of invading DNA, together the complex chops up that DNA, which kills the phage.


I'm passing fond of CRISPR because I wrote about it for my off-topic second year Ph.D. exam proposal
How all of this works. Source: My 2nd year topics exam proposal

My brief description doesn’t really do justice to the years of elegant experiments required to demonstrate how this process works. Much of the research was funded by the dairy industry because invading phages can totally DEVASTATE large-scale fermentations required for yogurt production and cheese-making.

Phages can TOTALLY screw up your breakfast
Phages can TOTALLY screw up your breakfast

Even though we have a pretty good grasp of how these systems recognize and chop up invading DNA, mysteries remain, including the fact that in many bacteria the CRISPR appears to be non-functional. However, I’m not here to probe the deep questions about CRISPR biology, I’m here to tell you about how we’ve harnessed this bacterial immune system for genome editing.


We took advantage of this part!  Source: My 2nd year qualifying exam proposal
We took advantage of this part!

It turns out, shockingly, that you can make a CRISPR system from scratch and get it to target any DNA sequence that you want. CRISPRs in bacterial genomes are made up of repeats interspersed with spacers that match different phages. However, it’s reasonably easy to change the spacers to match any stretch of DNA that your heart desires. CRISPRs cut DNA matching the spacer sequence, which is great for killing phages, and also allows us to delete almost any gene we want with remarkably high efficiency. However chopping up a genome into little bits is probably not the best way to edit out mutations to cure diseases. Luckily, scientists have figured out how to mess with the part of CRISPR that chews up the invading DNA. One group used a version of a CRISPR that doesn’t cut at all stuck to a fluorescent-GFP tag figure out exactly where different genes localized in living cells.

Source: Chen et al. Cell, 2013

In order to edit genomes researchers made versions of CRISPR that find a target DNA sequence, make a single cut, and then stop.


It looks like this. Source: Hsu et al. Cell, 2014
It looks like this.
Source: Hsu et al. Cell, 2014

Why would introducing a break in the DNA in a specific location allow us to edit genomes? The answer lies in how cells repair breaks in the DNA, which is a process called homologous recombination. This series of DNA gymnastics fixes broken DNA using an undamaged template.

Recombination is...complicated.
Recombination is complicated, and requires lots of sharpies

Normally that template is exactly the same as the broken DNA, so any mutations present in the original will still be there. However, the innovative idea behind genome editing with CRISPR is to force the cells to base their repairs upon a DIFFERENT stretch of DNA. The idea is to make a single cut inside a mutated gene, then have the cells repair the cut using an un-mutated, healthy template. Voila! Offending mutation erased, original function restored!

Source: Hsu et al. Cell, 2014
Source: Hsu et al. Cell, 2014


The idea is exciting, but we are certainly a few years away from being able to erase mutations inside patient cells. However I heard some exciting talks about ongoing efforts to improve the repair efficiency, change the cutting activity, and apply the system to different genetic disorders. I was particularly excited by Cecilia Cotta-Ramusinos’ talk about using CRISPR to correct mutations that cause sickle-cell anemia. Cecilia works at a biotech company named Editas, who are performing some thrilling research using CRISPR technology. The future looks bright!

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