February 18, 2017

Faster, Cheaper .. CRISPR

Let us imagine a quaint little colony where you and I live in peace but this peace is very often disturbed by outsiders who come in and dump garbage or play loud, ear-splitting music. What do we do? We collect photographs of the items that have created a nuisance in the past -- garbage bins, sound systems --  and store them in a photo album. We hire guards and give them  copies of photographs from this photo-album and ask them to check each and every visitor if he is carrying any of these unwanted items. When he finds a visitor who is carrying anything that matches with a picture in the album, he simply destroys the item or might even shoot the visitor. If a new culprit enters the premises, with a new disturbing item and is somehow overpowered by the community, then a picture of the new item is added to the album so that, in future, the guards can identify, destroy or deactivate it, if it is brought back again!
image from ScienceMag

This dramatised scenario is based on what happens when a streptococcus bacteria is attacked by a virus. Known hostile viruses, that are identified by a unique sequence of bases, or nucleotide molecules, in their DNA, are matched against a corresponding sequence in the bacteria’s DNA. Then a specific enzyme, the “gun”, is placed exactly on the matched part of the virus DNA and a “shot” is fired to break the virus DNA at the point of the match.  The virus repairs the DNA but the repair is never perfect. So the virus loses its original toxicity and the bacteria is saved from the viral infection. In the case of a new virus whose DNA signature is not available with the bacteria threatens the bacteria colony, some of the bacteria die, but those that survive keep a copy of the virus DNA signature in their own DNA, to be used against future attacks. To be technically correct, the virus DNA is not matched directly matched against its image stored in the bacterial DNA but against its complement, an RNA fragment, created from the DNA. This is like the neighbourhood guard being given a complementary negative of the image from the photo-album, not a photocopy.

This defence mechanism that bacteria have evolved over eons of evolution to defend themselves against hostile viruses is the backbone of a radical new technique called CRISPR/Cas9 that is sweeping swept through world of biotechnology and has revolutionised the way scientists modify the genes that define life and determine the characteristics of living organisms.

We have heard many horror stories about genetically modified (GMO) organisms but genes have been modified both naturally as well as artificially for many, many years. Random mutations or changes in genes happen naturally and they are propagated into progeny through the reproductive process while selective breeding of animals and plants are examples of artificial modification. But in all these cases, there is a lot of hit and trial involved and even when things work well, it needs multiple generations before the effect of the new genes become evident. Making similar changes directly, in a laboratory environment may be a little more easier, but not much. Current broad brush techniques are, slow, cumbersome, error prone and more often than not fail to achieve the desired goals. CRISPR/Cas9 promises to change this process so radically that it was widely tipped to win the Nobel Prize in 2016 but unfortunately it did not.

The genome, the sum total of all genetic material in any organism, is like a book  written with only 4 letters namely A, C, T, G. Actually each letter represents a base, an organic molecule. Specific sequences of letters form words. Some sequences of these words are irrelevant but other sequences of words form meaningful sentences that describe a specific recipe. In biological terms, a specific collection of meaningful sentences is called a gene and the recipe defines how the gene expresses or produces a specific protein. These protein molecules define how the living organism looks and behaves and their presence or absence can cause or prevent many diseases. The ultimate goal of genetic engineering is to alter these sequences of letters or bases in the genome of an organism so that beneficial outcomes, like disease resistance are enhanced and malicious outcomes like cancerous growth are inhibited.

But making these changes is not easy. The human genome can be viewed as a long chain of at least 3 billion letters -- spread over 23 chromosomes, or chapters, if we persist with the analogy of the genome being a recipe book. But only about 3 million of these are known to be a part of genes that play a definitive role, the rest are junk. To edit, or modify, an existing gene, any tool has to first locate its corresponding sequence of bases -- truly a needle in a haystack --  disrupt the sequence and then if possible replace it with another.

CRISPR -- Clustered Regularly Interspaced Short Palindromic Repeats -- are short identical sequences of bases that are located in the genome but are separated from each other by 32 unit sequences, called spacers, that are unique. These spacers, first located in bacteria that fight off invading viruses using the mechanism detected earlier, are images of various virus DNA that have attacked a bacteria in the past. Based on each such spacer DNA sequence, the cell itself, and now the scientist in a laboratory, can create an RNA fragment, called the gRNA, that will attach itself to a target DNA either in a virus, or in any other organism that the scientist wants to target -- precisely at the position where the sequence is identical to that in the original spacer. This is like walking down a street until you see a shop that is the shown in the photograph in your hand. Wherever, the gRNA stops, its sidekick, its  helper, a specific protein called Cas9 -- CRISPR associated protein 9 -- also stops, attaches itself, and like wire-cutter, makes a cut in the target DNA. This cut is repaired, but not perfectly, so the sequence of bases gets changed, the recipe becomes unreadable, the gene is disrupted and the corresponding protein cannot be produced. This two member team of gRNA molecule and Cas9 protein, that was earlier a defence mechanism for a naturally occurring bacteria is now a scientific tool that allows us to break a DNA at a very specific position in the chain.

Since the repair of the wire-cut DNA is imperfect, the gene is incapacitated or knocked-out. Very often this is desirable if the gene is responsible for some complicated disease. But what is even more interesting is if we can augment the two member gRNA, Cas9 team with a third member, an artificially prepared repair template that consists of a set of bases that we want to replace the original sequence with. Going back to our original analogy of the human guard, he is now given a bunch of flowers that he gives to the intruder after knocking out his garbage can and so instead of the stink of garbage, the intruder leaves our colony with the fragrance of roses.

CRISPR/Cas9 is a precision tool that can make small and precise changes in the DNA relatively easily. It is like using a thin brush to make changes in a precious painting instead of the earlier process of throwing a bucket of paint at it or using a high volume spray gun.

While CRISPR has been around since 1987, it was only in 2012 that Jennifer Doudna at the University of California, Berkeley and her collaborator Emmanuelle Charpentier  demonstrated the viability of using this two-molecule combination of gRNA and Cas9 protein to make precisions modifications on the genome. However in the same month, Feng Zhang of MIT’s Broad Institute filed for a patent for the same technology and the two teams have since been locked in an intellectual property battle of epic proportions. The commercial implications of this technology is immense. The race is now on to create specific gRNA molecules, that will locate and attach themselves to specific positions on the DNA of specific organisms, and the corresponding Cas proteins that will cut the DNA there. While human DNA is a very lucrative target as this may lead to cures of genetically transmitted diseases, even plant and other animal DNA is equally interesting as it would lead to disease resistant or high yield crops. All three principal actors in this drama have formed their own biotech firms to exploit the commercial benefits and Doudna and Zhang have already gone public. Finally, all three are widely tipped to win the Nobel Prize sooner or later for this remarkable technology.

Modifying the genetic code will lead to the creation of new, synthetic or hybrid organisms. This may or may not always be desirable but as we know,  there is no army that can stop an idea whose time has come and gene modification is one such unstoppable idea. Now we can do this faster, cheaper .. with CRISPR.