Reengineering Nature, hacking palindromes, turning yoghurt making into precision guided molecular missiles…

Image : Courtsey bostonbiotech.org

A palindrome is a word, phrase, number, or other sequence of characters which reads the same backward or forward. Simple examples are MUM, DAD, EYE, MADAM or a complex sentence like ‘Madam, in Eden I’m Adam’ could be read as palindrome.

Well english is not the only language with such sequences, the language of nature that all living things are written in also contains palindromes and other repeat structures. A palindromic sequence sequence in language of genetics is a sequence made up of nucleic acids within double helix of DNA and/or RNA that is the same when read from 5’ to 3’ on one strand and 3’ to 5’ on the other, complementary, strand.

5’ — G A A T T C — 3’
 3’ — C T T A A G — 5’

The palindromic sequences are famous as binding sites where various bacterial proteins called restriction enzyme (restriction endonucleases) bind to foreign DNA molecule for cleaving and rendering it ineffective. It is their cleavage feature that makes them an indispensible tools for genetic engineering. To date a plethora of such restriction enzymes have been discovered and are readily available as laboratory supplies and are extensively used as genetic scissors.

Dyed symmetry is another such symmetry in genetic sequence where two areas of a DNA molecule whose base pair sequence are repeats of each other, inverted relative to each other or are palindromes.

While studying the nucleotide sequence of gene responsible for alkaline phosphatase isoenzyme in Escherichia coli (bacteria in our guts) in 1987 at Osaka university in Japan, scientists discovered an unusual structure at the 3′ end flanking region of iap gene.

They found five highly homologous sequences of 29 nucleotides arranged as direct repeats interspaced with 32 nucleotides including a dyad symmetry of 14 base pairs as shown below. TGA is red marks the end of iap gene.

5′-[TGA]-ATGGGAG
 5′-CGGTTTA-[
TCCCCGC]-TGGC-[GCGGGGA]-ACTC-32 spaces 3′
 5′-CGGTTTA-[
TCCCCGC]-TGGC-[GCGGGGA]-ACTC-32 spaces 3′
 5′-CGGTTTA-[
TCCCCGC]-TGGC-[GCGGGGA]-ACTC-32 spaces 3′
 5′-CGGTTTA-[
TCCCCGC]-TGGC-[GCGGGGA]-ACTC-32 spaces 3′
 5′-CGGTTTA-[
TCCCCGC]-TGGC-[GCGGGGA]-ACTC-3′

Although these kind of structures were known to play a role in termination of transcription but Yoshizumi Ishino and his team had no idea what they stumbled upon.

As the technique advanced and more genomic data became available scientists discovered that these sequences were quite common in other species of bacteria. The sequences were formerly named in 2002 by Ruud Jansen as “clustered regularly interspaced short palindromic repeats” — CRISPR for short.

Ruud’s team also identified that these sequences were always accompanied by a collection of genes nearby. They called these genes Cas genes, for CRISPR-associated genes. The genes encoded enzymes that could cut DNA. Further sequencing revealed something peculiar about spacers between CRISPR sequences. The sequence resembled more with viral DNA.

What was the virus DNA doing in these sequences ?

Well the viruses are known to multiply by inserting their DNA into host DNA and use the host cell machinery to multiply as well as synthesize their packaging material. May be these genes were just relics of their past, a reminder of earlier encounters with viruses. But how good it would be for the organism if it did not use this information for its advantage.

An evolutionary genomics research group headed by Eugene Koonin had a similar idea, an idea that would one day turn into a versatile tool for biologists to engineer future of mankind, future of species that will form part of our biosphere.

The idea was that all along microbes would have been using these sequences as a defence mechanism against their parasitic invaders.

Thanks to the work done by scientists at Danisco, a DuPont probiotic company that we now understand how it works in bacteria. They found that microbes are using these genetic sequences to precisely identify and snip their host gene and render them in effective in the process.

In order to find virus resistant strains of bacteria to make yoghurt, team of scientists at Danisco inoculated bacteria with viruses. They not only isolated virus resistant bacteria in the process but also identified that it were the CRISPR sequences indeed that conferred resistance. The paper was published in Science in 2007 and marked a significant step towards understanding the underlying mecahnism which later on formed the basis of one of the modern biological tool for engineering organisms.

The CRISPR immune system works to protect bacteria from repeated viral attack via following steps:

  1. DNA from an invading virus is chopped into short snippets and those snippets are inserted into the CRISPR sequence as new spacers.
  2. CRISPR repeats and spacers in the bacterial DNA are transcribed i.e DNA is copied into RNA (ribonucleic acid).
  3. CRISPR RNA snippets bind to invading viral genome through complementary sequence using the CRISPR-associated enzyme and bacterial cell machinery.
  4. Endonucleases cleave the invading viral DNA at the target site leaving it in effective.

Bingo !!! The bacteria that mastered this technique are immune to viral attacks.

How did these tiny creatures achieve this feat using such a simple mechanism is beyond comprehension, well it won’t be called nature if it did not neatly tuck such secrets under its hoods.

So what’s the hack ?

Jennifer Doudna and her team got curious and they started unraveling the structure of Cas enzymes, they began to further understand the mechanism worked at molecular level.

The began to appreciate how bacteria were collecting samples of invading viral DNA, insertion it into their own genetic cassettes and engineering such versatile tools to cut and destroy the viral DNA.

They found that viruses were using this tool to feed the target coordinates to mount an attack. The molecular missiles loaded with crisper sequence are bound to hit their targets and destroy the enemy, all the bacteria had to do was to feed the right coordinates.

It was a light-bulb moment, the question was…

Can we feed this precision guided weapon any coordinates?

Who knows Doudna’s group may have stumbled upon a question that could put them in line for a nobel prize one day.

Doudna’s group eventually figured it out, although the answer did not come from yoghurt, it was stuck somewhere in their throats literally. It was the Streptococcus pyogenes, the bacteria that cause sore throat.

The figured out a way to supply Cas9 system in these bacteria with a snippet of RNA matching a sequence of target site DNA. The RNA molecule would than guide the Cas9 complex to the target site for it to make an incision.

Well their efforts finally paid off and a precision cut molecular missile was invented, the missile is programmable to make a cut at desired site, this was begining of new era in gene editing.

This technology has widespread applications, thousands of papers have been published demonstrating use of the technique in silencing genes, replacing faulty ones, precision editing story and destiny engrained in DNA of somatic cells as well as germ lines in many life forms including us.

It sure has much wider implications, be it in correcting our genetic mistakes, finding cures for diseases, strengthening our defences against invading pathogen, improving efficiency of our food production systems, engineering our ecosystems so that we can continue to inhabit the planet despite the mess we have already created here, nudging our biology to become super humans or simply tinkering biology to jump its underlying fragile chemical substrate and become immortals.

Sky is not the limit at more, the technique is already here , the raw materials and equipment is widely available, with right kind of know how one can effectively order online a pre configured tool to precisely cut a piece of DNA and fix an organism in a makeshift biology lab in the garage. So it is really up to us what we do with it, weapon is in our hands how wisely we use it will determine not only our future on this planet but planet’s future as well. Its reins are in our hands.

As per the prestigious scientific publication Nature calls it is the disrupter, a powerful gene-editing technology, the biggest game changer to hit biology since PCR, with huge potential but its use comes with pressing concerns.


Originally published at scriptgrandeur.wordpress.com on January 10, 2016.

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