International MPN News, Science & Opinion

CRISPR/Cas9: Gene editing with precision

 This changes everything

Jennifer Doudna

Jennifer Doudna

Emmanuelle Charpentier

Emmanuelle Charpentier

Dr. Jennifer Doudna of the University of California at Berkeley and the Howard Hughes Medical Institute, and Dr. Emmanuelle Charpentier, of the Hannover Medical School and Helmholtz Centre for Infection Research (HZI), Germany and The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umea University, Sweden…. Their collaboration led to the discovery of a new method for precisely manipulating genetic information in ways that should produce new insights in health and disease, and may lead to the discovery of new targets for drug development.
[There’s a short video of these two primary discovers of CRISPR Cas gene editing system here.]

  Finally … a means to correct genetic errors easily and precisely.

Dr. Mirthra Ravindar LabTalk

Dr. Namritha Ravinder LabTalk

 Using CRISPR, MIT researchers reported curing mice of a rare liver disorder. the first demonstration that CRISPR can reverse disease symptoms in living animals

There is need in the field for a technology that allows precise targeting of nuclease activity (or other protein activities) to distinct locations within a target DNA in a manner that does not require the design of a new protein for each new target sequence. In addition, there is a need in the art for methods of controlling gene expression with minimal off-target effects….

The present disclosureCRISPR patent 1 provides a DNA-targeting RNA that comprises a targeting sequence and, together with a modifying polypeptide, provides for site-specific modification of a target DNA and/or a polypeptide associated with the target DNA. The present disclosure further provides site-specific modifying polypeptides. The present disclosure further provides methods of site-specific modification of a target DNA and/or a polypeptide associated with the target DNA CRISPR patent 2The present disclosure provides methods of modulating transcription of a target nucleic acid in a target cell, generally involving contacting the target nucleic acid with an enzymatically inactive Cas9 polypeptide and a DNA-targeting RNA. Kits and compositions for carrying out the methods are also provided. The present disclosure provides genetically modified cells that produce Cas9; and Cas9 transgenic non-human multicellular organisms.”

– From Patent Application 13/832849  (click image to enlarge)


My introduction to CRISPR was a result of a nasty habit: Taking that first cup of coffee back to bed and listening to the Morning Edition news on NPR.   Joe Palka was reporting on gene editing, a new tool geneticists were using,  and the work of a potential Nobel laureate up on the Berkely campus. Her name:  Jennifer Doudna. Gene editing:  Could we actually edit out the JAK 2 mutation? The MPL, CALR or myriad other genetic confusions that gave rise to our myeloproliferative neoplasms?

The foundation has already been set and animal trials completed.  Using CRISPR, MIT researchers reported curing mice of a rare liver disorder. the first demonstration that CRISPR can reverse disease symptoms in living animals.

The MIT researchers delivered the CRISPR package that includes RNA guides, the Cas 9 gene (see below) and a DNA template containing the correct sequence of the mutated gene.

The results, reported in  Nature Biotechnology, March, 30, 2014 and Phys.Org: “Using this approach, the correct gene was inserted in about one of every 250 hepatocytes—the cells that make up most of the liver. Over the next 30 days, those healthy cells began to proliferate and replace diseased liver cells, eventually accounting for about one-third of all hepatocytes. This was enough to cure the disease, allowing the mice to survive ….”

To understand the newest, most promising molecular biology technique – gene editing via CRISPR Cas – we have to take a look way back. To the very beginnings of life on Earth.

Duplication and the rise of life

However life began on Earth, the only certainty is it had to have had the ability to reproduce itself. The early stew of  gases and chemicals could produce organic molecules that might polymerize into macromolecules. But the whole business needed a system to reproduce itself… or just fizzle out.


Source: Smart Therapeutics

Lacking that ability, it would simply randomly combine and fall and disappear as innumerable chemical combinations were doing in that steamy prebiotic neighborhood.  Fortunately, four of those chemicals,  nitrogenous bases, had the peculiar quality of pairing only with each other: Adenine (A) pairs ONLY with Uracil (or Thymine, in DNA)  (U) and Guanine pairs ONLY with Cytosine (C)

The affinity of this handful to link only to each other is the root of the double helix two stranded DNA that encodes our own multi-cellular, warring, loving, complex existence.  In the beginning,  that string of nucleic acids — and a covering that could pass for an overcoat until something better came along —  was enough  to create some elements of life..

Those basic building blocks seeking out specific matched bases, permitted RNA to produce an enduring and identifiable molecule, one that under favorable conditions  could reproduce itself.

It may not have been life, but it was a pretty good place holder for several hundred million years or so until bacteria emerged.

Why viruses don’t rule the world Or…why bacteria are still around.

In that great tidewater basin of time, the billions of years between a cooling mineral, gaseous planet Earth and the rise of life, in a time before oxygen, there were viruses.

Mindless,  sexless, able to live without eating or breathing, depending on other cells for reproduction, parasitic viruses are on the cusp between life and non-life.

Their strand of DNA code, their capsid protein shell to protect its genome and uncanny ability to appear as nutrients to living cells was enough to sustain their generations.

One of the earliest struggles for survival on Earth was the battle to the death between viruses and the bacteria they would infectThe defensive weapon manufactured by bacteria provided the blueprint used to develop CRISPR Cas, an efficient, powerful tool to edit sequences in the human genome.

It wasn’t humans who figured out how. A microbe  with no brain and a single cell figured it out.

Both viruses and bacteria may have been on Earth for over 3 billion years. Some say bacteria didn’t appear until  oxygen was first available on Earth, about 1.5 billion years ago.  But no matter. Viruses, like pebbles underfoot, are in no hurry, are never patient nor impatient.  100,000 of them could shelter under a grain of sand for millennia, waiting. Waiting, it seems, for bacteria.

The arrival of bacteria, single-celled microbes, presented a ready source of food and reproduction to the mindless, undead virus.

Bacteria are single celled microbes. The cell structure is simple as there is no nucleus or subunits wrapped in membranes.  All their genetic information is contained in a single loop of DNA. Some bacteria have  extra genes to produce a useful protein but pretty much that’s the architecture of a bacterium.

And their survival is an essential step in human existence. There are 10 times as many bacteria in the human body – mostly the gut – than there are human cells in tissues and organs.

On the strictly cellular plane, we are more bacteria than human.

Question is how did they survive the attack of the much more plentiful, well established, invisible viruses?

The bacterial defense system is primitive and effective. In a virus attack, after the virus penetrated its cell wall, the bacterium would snatch a tiny segment of its DNA. These few base pairs of virus DNA would be interrogated, duplicated and stored Once the attacker was identified, the bacteria could mobilize its own immune system and use enzymes guided by RNA derived from the virus’s transcribed code to cut the virus’ DNA and stop further reproduction.

Cut may be the wrong word, although that is the effect.

e.coli at 10,000x magnification

e.coli at 10,000x magnification

It’s more of a chemical process, mobilizing proteins to bind the DNA and enzymes to eat through the virus’ DNA strands like acid, in effect castrating the virus by deleting a key section of code that made up its reproductive chain.

Beyond assuring its own evolution and the ultimate emergence of complex and equally murderous creatures, this powerful defensive action has become the model for gene editing, sophisticated manipulation of disease at the molecular level   If bacteria can do it, why could we not leash the same cut and paste precision technology to a pair of genomic scissors to serve the human genome?.

We did appropriate the  technology by mobilizing the palindrome.
Madam, I’m Adam.

Probably you’ve seen that construction before. Madam I’m Adam reads the same backward or forwards.  Like: Dammit, I’m mad! It’s a palindrome, or a palindromic sequence.

The palindrome has a special use in molecular biology. Paired strands of nucelotides running in opposite directions make up the DNA double helix.  (A single strand is palindromic if its complement, nucleotide by nucleotide, is its reverse.) The molecular palindrome is used by restriction enzymes to recognize and cut DNA sequences.  For example, one restriction enzyme called Eco-R-one (EcoR1), isolated from E.coli bacteria, recognizes and cuts a specific sequence of DNA base pairs.

The four letters that make up the DNA book are G (Guanine), A (Adenine) T (Thymine) and C (Cytosine) with U (Uracil) standing in for Thymine in RNA sequences.   These bases always pair the same way:  G with C and A with T.  Here’s a recognition sequence:

 G A  A  T  T  C

C T  T  A  A  G

The top strand reads GAATTC while the bottom strand reads CTTAAG. If the DNA strand is flipped over, the sequences are exactly the same ((GAATTC and CTTAAG).  The restriction enzyme EcoR1 recognizes the GAATTC sequence.

And here’s how Eco-R-One would cut the sequence.,


Source Recognition Sequence Cut
EcoR1 Escherichia coli 5’GAATTC3’CTTAAG

5′—G     AATTC—3′

3′—CTTAA     G—5′

(Example courtesy of Wikipedia)

Bacteria knew nothing about any of this, not palindromes, not base pairs and definitely not double helixes. And yet they used the recognition system of a scrap of virus DNA  to find and cut the sequence that permitted the virus to reproduce.

Once we understood this process, we were on our way to appropriating that same technology to target and cut, silence, up-regulate, delete or add a sequence of our own to a strand of DNA. We’ve already done it successfully in zebrafish, in e-coli, in mice.

The Cascade and the Palindrome

The palindrome is at the heart of the CRISPR Cas/9 name and technique:  Clustered Regularly Interspersed Short Palindromic Repeats. Some other terms specific to CRISPR are:

Cas= CRISPR-ASsociated. There are various functional Cas proteins. (Cas9 is a killer, the neutralizer.)

PAM = Protospacer Adjacent Motif.   This is the targeting bullseye, those DNA sites on either side of the base pairs – or single nucleosides – identifying the target signature.

CASCADE is the loaded mother ship.   Cascade = CRISPER-ASsociated Complex for Anti-viral Defense. This is the bundled tool that includes CRISPR RNA guides to glom onto the viral target.  RNA, with its affinity for only a specific sequence of amino acids, is an infallible homing tool. Cas proteins aboard CASCADE  unwind the viral DNA and search for the PAM and another Cas protein finds the matched sequence and the Cas9 protein zaps it.

In cartoon action, here’s how the e.coli bacteria mounts an anti-viral CASCADE  consisting of five Cas proteins plus the CRISPR RNA (crRNA). This crRNA was created out of the short segments of viral DNA, translated and then turned into a tool to hone in on the viral target via that short sequence identified by PAMs.

The Cascade binds to target DNA sites then signals the attached Cas proteins to cut the bound DNA ending its proliferation.

The CRISPR Cas9  summary

“There are two main components of the system says Dr. Namritha Ravinder,  of Life Technologies. “… There’s the Cap 9 nuclease that acts as a DNA cleavage enzyme…

  Just by itself it doesn’t know where to go within the genome… second part is the guide RNA which has two pieces in it  The CRISPR RNA ,which defines target specificity, anywhere from 17 BP to 80 base pairs and the tracRNA component and that stays constant wCRISPR Cas9 SLIDECRISPR Genome editing made Easyithin the host.  The guide RNA forms a compound with Cas9 protein and this whole unit is recluded to the genomic locus within the DNA just by base pairing the RNA sequence and the target sequence within the genome.     So this base pairing tells the system this is where I need to bind and this is where I need to cleave…

“CRISPR Cas9 as transformative… This group targeted five different genes within stem cells using 5 different guide RNAs one for each gene within the genome. And what they could do is target all at the same time and pick clones downstream and find clones that all the five genes knocked down in the system.

“It’s easy to design, very simple…Wide range of applications: generate disease models.. edit stem cells for gene therapy..”

JAK2 V617F…are you listening? We’re loading up the Cascade and coming to get you.

 –Zhenya Senyak

Take me back to the Contents

©  2014, MPNforum, LLC and Zhenya Senyak. Unauthorized use and/or duplication of this material without express and written permission is prohibited. Excerpts and links may be used, provided that full and clear credit is given to with appropriate and specific direction to the original content. All rights reserved under Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License.

Comments on: "CRISPR/Cas9: Gene editing with precision" (8)

  1. Great work again Zhen , you never disappoint with your up to date reports and give all us patients hope for the future.

  2. Mercedes Maharis MA MS MA said:

    So, how far away are clinical trials… and what method does the gene therapy take?

    • For us with MF, Mercedes when is the critical question.
      CRISPR/Cas is being used right now in animal studies in labs. Clinical use in MPNs is anyone’s guess. The JAK2 mutation was discovered in 2005 and the Jakafi was approved by the FDA at the very end of 2011. Since CRISPR/Cas is a technique and not an investigative new drug, the path to clinical use can be much faster. We’re hoping to come back from ASH with a much clear idea of timelines., In any case, the ability to replace a mutated gene is dependent, in large part, on the identification of that gene.

      As to method of delivery etc. let me refer you back to the article and the link to, below describing the MIT animal study reversing a liver disorder using CRISPER/Cas9. There is a wealth of detail their and quotes from the senior MIT scientist Dr. Daniel Anderson.

      In experiments with adult mice carrying the mutated form of the FAH enzyme, the researchers delivered RNA guide strands along with the gene for Cas9 and a 199-nucleotide DNA template that includes the correct sequence of the mutated FAH gene.
      Using this approach, the correct gene was inserted in about one of every 250 hepatocytes—the cells that make up most of the liver. Over the next 30 days, those healthy cells began to proliferate and replace diseased liver cells, eventually accounting for about one-third of all hepatocytes. This was enough to cure the disease, allowing the mice to survive after being taken off the NCTB drug.

  3. Ron Russell said:

    “Finally … a means to correct genetic errors easily and precisely.” Please! This is so overstated, overwritten and unfocused that it defies comprehension. We need basic info on what might offer us some help. We don’t need a rambling lesson in science and semantics. I suggest a serious revision that focuses solely and clearly on this research and how it might lead to some benefits.

    • Sorry you had difficulty comprehending this article, Ron. Could be a left brain/right brain thing. We do aim to reach those with either brain preference… although it’s hard to reach everyone. Feel free to skip the science and the history and go straight for the bottom line…although I don’t see how you would understand CASCADE or the palindrome itself without some minimal intro. Both the direct benefits and the straightforward research are in the article. See Dr. Namritha Ravindar‘s full presentation, read the patent application, read the reference to the MIT research and the Nature Biotechnology Report, the Joe Palco transcript, watch the CAS9 video. It’s all there in the article. You just have to click on some of those red things (we call them links) to get to the underlying reports. When you finally realize this is revolutionary and a highly precise, efficient and economic means to correct genetic mutations you might be less exercised at our enthusiasm.

  4. I agree Pam, absolutely. This intro was filled with scientific and historic stuff that may have blurred the true significance of the breakthrough. A fast, easy way to snip out multiple mutations in one payload and cut and paste in healthy replacements is a critically important, game-changing approach to MPN treatment. OK, we’re some distance from clinical application… and there are many competing genetic clonal disorders. But we’re clearly on our way… and we have a powerful international crew of MPN scientists and specialists who will start using CRISPR for MPNs. “This is a revolution happening right before our eyes,” is the way one respected expert put it.

  5. Pam Coopersmith said:


  6. Bonnie Kaye Evans said:

    Thanks for making me go back and read this article. I sure hope the can make all MPNs dissolve away.

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