Why is everyone talking about CRISPR-Cas9 technology?

For the first time, people outside scientific community are actually curious about snipping DNA using CRISPR-Cas9 technology. You must be wondering why on earth is everyone talking about CRISPR-Cas9 technology.

The reason everyone is excited about CRISPR-Cas9 is that the snipping of DNA can (potentially) do lots of things:

  • cure human diseases (cancer, Alzheimer’s etc)
  • increase crop yield (more nutritious and high-yield rice)
  • limit mosquito population (no malaria fever)
  • produce hornless dairy cows (you can hug your cow without fear!)

and the list is endless.

But how can CRISPR-Cas9 do so many amazing things ?

The answer lies in the ability of CRISPR-Cas9 technology to change the DNA sequence.

Our body is made up of trillions of tiny building blocks called ‘cells’. Each of these cells contain DNA molecules which decide the identity and health of each cell (and the organism). The DNA molecule is defined on the basis of how the four letters A, G, C, and T are arranged. Our DNA is approximately 3.2 billion-letters long, and if you could slice out some part of the human DNA and read the sequence, it might look something like this:

GCGTCCGAGGTAGACGATGG

Humans are humans because we have different DNA sequence than monkeys, plants, pigs, or ostrich. When I say different DNA, what I mean is that if you read the DNA sequence, let’s say a random region of the DNA, it would be arranged slightly differently in each organism. For example, a 20-letters region somewhere in the vast expanse of DNA molecule might look like the following:

GCGTCCGAGGTAGACGATGG      human

GCGTCCGAGGTAGACGATCG       monkey

GCGTCCTTCCTAGACGATGG        rice plant

This is just a hypothetical example and shows sequence variation in just a tiny 20-letters fragment of DNA in different organisms. Can you imagine how differently 3,200,000,000-letters long DNA can be arranged? Countless ways (your computer might crash if it starts guessing different combinations/arrangements of the four letters in 3.2billion-letters long  DNA).

Similarly, the DNA sequence is altered in different disease conditions. For example, DNA from brain cells of healthy person versus brain tumour patient (hypothetical example) might look like this:

GCGTCCGAGGTAGACGATGG         healthy person

GCGTCCGAGGTAGACGATGAG      brain tumour patient

This extra ‘A’ in the brain tumour patient DNA might seem as a tiny error to human eye but it can change the behaviour of brain cells to divide continuously, giving rise to the tumour. Theoretically, if we can revert back the DNA sequence to its original healthy sequence, we can transform tumour cells to normal cells.

And that’s where CRISPR-Cas9 comes into the picture. Yes, it can perform this magic for us. The Cas9 protein (the molecular scissors) can be directed to this particular faulty part of the DNA, where it can cut the DNA, slice out the extra ‘A’, and then cellular machinery can re-join broken ends. The CRISPR Cas9-mediated gene-correction method can be used to virtually treat any disease. Similarly, suppose if we know, adding extra ‘GCTG’ to rice DNA could make it more nutritious or produce longer grains, we can do that using CRISPR-Cas9 method.

GCGTCCTTCCTAGACGATGG                  normal rice plant

GCGTCCTTCCTAGACGCTGGATGG       rice plant with longer grains

Again, this is just a hypothetical example. Rice DNA is 3,000,0,000-letters long and many more changes in the DNA sequence would be required to produce longer-grain variety. Researchers around the world studying rice DNA will one day find out what changes in DNA sequence are required and then using CRISPR-Cas9, we can introduce those changes into rice DNA and relish delicious Hyderabadi Biryani .

At the molecular level, CRISPR-Cas9 system consists of the following components (donor DNA is optional and is needed when you want to make a specific change):

1. Cas9 enzyme: a protein that has the ability to cut/snip DNA. The first step in DNA sequence correction is making a double-stranded break (DSB) at the desired target site. Once a break is made, DNA repair proteins gather at the broken ends of DNA and ponder how to re-join the broke ends.

2. guide RNA: a small piece (20-letters) of RNA that tells Cas9 where to cut in the long DNA molecule. Therefore, guide RNA has the information about target DNA sequence. Guide RNA sequence is essentially similar to target DNA except for the fact that it has a different letter ‘U’ instead of ‘T’.  For example,

GCGTCCGAGGTAGACGATGG            DNA target sequence

GCGUCCGAGGUAGACGAUGG          guide RNA sequence

As you might have guessed, CRISPR-Cas9 works as a Cas9/guide RNA complex, wherein guide RNA has the information about where to cut and Cas9 has the ability to cut. Scientists, express or deliver Cas9/guide RNA complex in the cells and deliberately make a cut at the  faulty sites in the DNA.

3. donor DNA: The donor DNA has the corrected DNA sequence. The repair enzymes use donor DNA to re-join broken ends, and the end result is DNA sequence similar to desired ‘healthy’ DNA.

I hope you now have an idea of how Cas9/guide RNA scissors work. Of course, this is just a layman description of how it works and there are many intricate technological challenges that need to be addressed before we try CRISPR-Cas9 gene correction on humans. But we are making progress and not very far from doing ‘seemingly impossible things’ in all aspects of life around us using CRISPR-Cas9 magic !

NgAgo: Genome-editing by a DNA-guided enzyme

      While much of the scientific community was very busy optimising CRISPR-Cas9 technology for their affectionate model systems (mammalian cells, plants, organisms- mouse, zebrafish), a recent article in Nature Biotechnology has left everyone baffled.

The article was published online ahead of print in Nature Biotechnology journal last month, authored by Chunyu Han lab members from China. The authors demonstrate that a protein of Argonaute class can be utilised to edit genome using a DNA guide sequence. The enzyme belongs to the bacterium Natronobacterium gregoryi and has been christened  as NgAgo (Natronobacterium gregoryi Argonaute). By the way, I like rhyming NgAgo with San Diego, so if you are writing a rap song about genome-editing, just give it a thought!

But, seriously, why so much buzz about an enzyme that is guided by a  24-nt DNA sequence rather than 20-nt RNA sequence as in CRISPR-Cas9 system?

Well, it appears that NgAgo system has many advantages over Cas9 system.

  • DNA synthesis of 24-nt guide DNA (gDNA) is cheaper than RNA synthesis. Also, RNA is a very delicate and less-stable molecule requiring more attention when setting up an experiment.
  • NgAgo system is simpler and requires just the 24-nt guide DNA that is modified at 5′ end (phosphorylated, companies can do that for just a few extra bucks). On the other hand, Cas9 system requires 20-nt guide RNA (gRNA) plus a longer tracrRNA molecule (around 70-nt long). Although, the two RNA molecules of Cas9 system can be expressed together as fusion single guide RNA (sgRNA). But that requires cloning of gRNA into a plasmid (phew cloning) or in vitro synthesis (even worse for postdoc/PhD students!).
  •  Target site recognition does not require PAM (3-letters NGG) or any other sequence of that kind. This makes life easier for researcher to virtually target any part of the genome without bothering about the availability of PAM sequence. All you have to do is copy 24-nt DNA sequence from your desired locus and send it to an oligo company. No need to use a gRNA prediction tool. Also, some parts of the genome (such as at the start and end of the gene) are AT-rich and you might be unlucky to find NGG, making it difficult to design gRNA for Cas9 system for such regions.
  • NgAgo system is powerful system to edit GC-rich regions. Authors have shown in the article that NgAgo system can penetrate GC-rich, obscure regions of genome and outperforms Cas9 system which can hardly make any impression there. This might have more to do with guide loading onto enzyme: gRNA with high GC content are prone to self-annealing and form secondary structure before they are loaded onto Cas9 enzyme.
  • NgAgo system is more stringent and cannot tolerate mismatches in gDNA/target DNA binding. Making a cut at undesired sites (off targets) is a big issue when it comes to gene therapy (and molecular biology in general). This article highlights the fact that NgAgo system does not like mismatches in guide DNA sequence (a single mismatch out of 24-letters can compromise cutting activity whereas 3-letters mismatch completely abolishes enzyme’s ability to target a DNA sequence). Cas9 can tolerate up to 4-5 mismatches and will cut off-targets as well. This off-target issue might not seem very important for regular genome-editing of completely messed up cancer-cell lines but could be a decisive factor when thinking about editing genome in a patient (we are far away from that).

Before Cas9 supporters hit me , let me tell you that everything is not good about NgAgo and there are some Cas9 moments as well.

  • gRNA loading onto Cas9 can occur at room temperature (in vitro) as well as physiological temperature (37 0C) without affecting Cas9 activity. NgAgo is more fussy when it comes to gDNA loading:
    1. in vitro loading: If you want to use recombinant, pre-assembled NgAgo/gDNA for transfections, you will have to incubate NgAgo plus gDNA at 55 0C for 1 hour. This weakens enzyme which can no longer make a double-stranded break but can only make a nick (cuts one strand). A nick is not very useful for genome-editing because it gets repaired by cells ‘repair machinery’ without leaving any changes at cut-site.
    2. in vivo loading: I am not sure if one can express gDNA (single-stranded DNA) in mammalian cells, and that too 5′ phosophorylated ! Therefore, you will have to transfect cells with NgAgo-expression plasmid plus gDNA. You must be wondering do I have to boil cells at 55 0C for 1 hour for gDNA loading? Good news is that your cells do not have to go through such ordeal. It appears that gDNA can be loaded onto NgAgo enzyme  when the protein is being freshly translated/expressed.
    3. CRISPR-Cas9 is powerful tool for gene activation/suppression, tracking etc. However, we must remember that it took 3-4 years for Cas9 to celebrate its success in wide applications. No doubt that the NgAgo system can be engineered to do the same.
  • Genome-wide studies have shown that after all Cas9 is not that bad when it comes to cutting at off-targets. Only head-to-head genome-wide studies in future will tell if NgAgo is actually better than Cas9 in terms of off-targets.
  • Authors have shown that NgAgo-mediated indel frequency for many tested genes is 20-40%. Cas9 system was not tested in parallel for those genes. I would assume that Cas9 is more potent tool for gene disruption because many studies have reported up to 60-70% indel frequencies.

Will NgAgo overshadow CRISPR-Cas9?

Hold on guys, give it some time. NgAgo is just one article and a month old. It surely appears cost-effective and simpler but needs to be scrupulously tested head-to-head with Cas9 system. The biggest concern with NgAgo is the impossible task of expressing 5′ phosphorylated single-stranded guide DNA in cells. Cas9 guide RNA enjoys various methods of delivery: in vitro pre-assembly, plasmid-mediated co-expression, virus-mediated expression etc. But I am sure scientists will come up with better variants of NgAgo or who knows, altogether new class of guided-endoncleases!

We have come a long way- since the discovery of DNA as the genetic material to mapping of the human genome (3,200,000,000-letters) and now to editing any letter in the human genome. The last 10 years have seen so much progress in the field of genome-editing. The RNA guide-based system, CRISPR-Cas9, has revolutionised genome-editing area and made us more ambitious to target many more genes and organisms in less time and money. Future studies will unleash the potential of NgAgo system and show whether it can overtake powerful CRISPR-Cas9.

 

Genome-editing. Why? How?

Genome-editing. Why? How?

Want to know how scientists edit (cut some part, paste something new) the blueprint of life- DNA?

Well, you are at the right place. This blog is all about DNA molecules in each of our cells (except red blood cells, these guys live without DNA!) and how molecular biologists alter a little information in long piece of DNA to make life better.

This might take some weeks to fill in more information, so please bear up with the delay and visit again.