CRISPR in your Future

CRISPR is a two-year old technology developed at Berkeley, Harvard Stem Cell Inst and elsewhere, that is making genetic engineering faster, simpler, and more accurate in the lab.  Last year, they figured out how to insert and delete genes.  This year there are methods for repressing and perhaps promoting genes (epigenetically, without modifying the genome) using CRISPR-derived technology.  Enthusiasts say they will soon be able to turn genes on and off at will.  It is my belief (I’m not alone) that aging is controlled largely by epigenetics—what genes are turned on, when, and where.  Rapid progress is being made identifying the genes that need to be promoted and the genes that need to be repressed to restore an older person to younger gene expression.  It may be that by the time we are ready with this knowledge, CRISPR will be ready to implement it in living patients. The biggest question mark at this early stage is delivery.  How do you get the CRISPR protein/RNA complex into the cell nucleus?  

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The first generation of genetic engineering was turned to therapeutic use by means of genetically-modified viruses.  Viruses already know how to drill their way into a cell wall, find their way into the nucleus, then copy their own DNA into the chromosomes that they find there.  For therapeutic applications, first a replacement for a defective gene is added to the viral DNA, so that when the virus copies itself into the host DNA, the therapeutic gene will be copied along with it.  Second, the virus is denatured, crippled so that it has a limited lifetime in the host, and won’t keep multiplying at the host’s expense.  (The host is the patient.)

First-generation gene therapies are crude in that there is no ability to control where in the genome the therapeutic gene is inserted, or to turn it on or off.  Adenoviruses replaced the lentiviruses used in early trials because at least they insert the gene in the same place on the same chromosome. Results have been mixed, unexpected side-effects are common, and gene therapies have been considered only for patients with life-threatening conditions.  Nevertheless, there are about 2,000 clinical trials currently approved world-wide.

Zinc finger nucleases and TALEN are second-generation technology.  These are enzymes that contain a protein-based portion which can be engineered to bind to a specific segment of DNA, plus a snipper enzyme that cleaves DNA (both strands) where it binds.  Potentially, a gene can then be removed or inserted.  The principal disadvantages are that they are time-consuming and therefore expensive.  It is not easy to engineer a protein that reliably binds to a particular target stretch of DNA.

CRISPR technology is a candidate for third-generation gene therapy, based on a DNA-splicing protein that evolved in bacteria as a defense against invading viruses.  Viruses (bacteriophages) can infect bacteria and insert their own viral genes into the bacteria’s genome.  CRISPR-associated system protein (called Cas9 enzyme) splices the DNA at just the right place to remove the virus, restoring the integrity of the bacterial DNA.

This nifty defense evolved in bacteria and archaea, but not in animals or plants.  Now, researchers have figured out how to lift the Cas9 enzyme and the template that guides it, modify the template at will, and inject it into the cell of a human or lab animal.

(The acronym stands for Clustered Regulary-Interspaced Short Palindromic Repeats.  What that means, and why there should be little palindromes spread through bacterial DNA are questions for another day, because they don’t really help understand how CRISPR works, its potential and its limitations.)

The big new advantage is in the Guide RNA (gRNA), which can easily be sequenced to match (as a complement) any short stretch of DNA in the genome of a human or test animal.  The Cas9 splicing enzyme then finds the spot that matches the complement of the gRNA, and that’s were it does its job.  Curiously, the gRNA is not targeted as reliably as zinc finger or TALEN, and occasionally latches on to a stretch of DNA that is a near-match, so a gene can be inserted or a chromosome cleaved at the wrong place. One solution to this problem that is being tried is to prepare two gRNAs for the same stretch of two strands of the double helix, and to modify the Cas enzyme so that it only cleaves the DNA if both strands are struck simultaneously.

 

CRISPRi

CRISPR techniques can be adapted for epigenetic control, not cleaving a gene at all, not modifying the DNA permanently, but silencing a gene that we may wish to turn off.  (The “i” is for “interference” and the acronym is intended to be reminiscent of RNAi, or RNA interference, which is another second-generation technology, useful for silencing genes only.)  With CRISPRi, tags are attached to the DNA at a target location such that they interfere with transcription of a gene in progress.  Potentially, CRISPR can be adapted to promote genes as well, but this is more challenging.  It is in the promise of full epigenetic control that the most exciting applications lie, in my opinion.

 

Delivery

This is one of the big issues remaining before CRISPR technology can become a useful therapy. So far, it has been used on cells in culture. It has also been delivered intravenously at high pressure to lab mice, but the therapy only reaches a small proportion of cells.  It can be micro-injected into the cell nucleus, but this is practical only for experiments, one cell at a time.  CRISPR kits are being sold as plasmids, which is their original progeny in bacteria.  Plasmids are small loops of DNA, commonly exchanged by bacteria, but foreign to animal and plant cells.  There are papers describing adenovirus applications that combine with CRISPR to offer both control and penetration, and these are so far in early demonstration stages.

 

Active and Inactive DNA

Sewing thread is made of multiple, tiny fibers twisted together.  The twisted structure has an integrity of its own, but it’s liable to become tangled and knotted, so we keep it wound neatly on a spool until we need it.  The cell does the same thing with its DNA.  The twisted structure is the double helix.  And the DNA strand is so long that it’s liable to become tangled.  (We have about a 6-foot length of DNA in every cell, stored in a nucleus that is less than a thousandth of an inch across.)  The spools are protein molecules called histones, and threads of DNA are wound around them for orderly storage.  Each chromosome is a continuous thread of DNA, and there are many spools along its length.  At any given time, some parts of the thread are open and available, while other parts are tightly-spooled and hidden from chemical activity. Tightly-spooled DNA is called heterochromatin, and it is inactive, not available to be transcribed into proteins.  Unspooled DNA is euchromatin, and this is the active form of DNA, ready to be transcribed.

So what happens if a CRISPR unit (a Cas enzyme) comes along that is targeted to a part of the DNA that’s tightly wound up as heterochromatin?  Not much happens.  The CRISPR process is much less efficient on heterchromatin compared to euchromatin.  Imagine a reader scanning through a book looking for a particular phrase.  The process is much more likely to work if the book is open.  This is another challenge for realizing the potential of CRISPR.

 

Which genes to turn on and turn off?

I wrote a series of blog posts on this question last year.

Hormones that we lose as we age include melatonin, thyroxine, DHEA and (recently announced) GDF11

Hormones that are overexpressed, and we need to repress or block include NFκB, TGF-β and (recently announced) JAK/STAT signals

 

The Right Technology for Anti-Aging Remedies

I’m not ready to have my genes replaced, thank you very much.  I think that there are genes that are associated with longevity, and several together might add a decade or more to life expectancy.  But replacing genes is permanent, and it’s based on a technology fraught with unexpected side-effects.  Besides, my body already knows how to be young.  When it was young, it had the same genes it had now, but the epigenetics—the set of genes turned on and off was somewhat different.  I’m willing to bet that restoring a young epigenetic state to my same old genes will make me young, and that’s why I’m pumped about the CRISPR technology.

 

Read more:

Fore-Cas-t from The Scientist
Kurzweil AI on CRISPR gene therapy
Cas9 as a Versatile Tool for Engineering Biology
Comparison of Zinc Finger, TALEN, and CRISPR
Adenoviral vector delivery ofRNA-guided CRISPR
CRISPR-Cas systems for editing, regulating and targeting genomes
CRISPRi explained at a technical level
On-line discussion of speculating about use of CRISPR as a gene promoter

12 thoughts on “CRISPR in your Future

  1. Now you are beginning to sound like Kurtzweil (and that’s not a good thing to me). Using CRISPR to regulate gene expression in all of our cells, stem cells most importantly, to control the very divergent transcription patterns of the hundreds of cell types that constitute the body? I think not. And it would be easy to see how CRISPR might bind to correct sequences, but then how would you control CRISPR so it allows the right temporal patterns of transcription and modulate, rather than turn on or off the amount of transcription of specific life-stage related genes? There is no mechanism I know of,, and those patterns would be different in different tissues, so you’d have different CRISPRs in different tissues and contolled how? By signals in the blood perhaps?
    The techniques to change the transcription patterns to youthful profiles by substances in the blood is already here and won’t require the century that CRISPR introduction into cells, then into the nucleus and all the different cell types and then control of transcription rates, honestly? Of course you mention some downstream players, DHEA, (you didn’t include Klotho), GDF11, but do you know that controlling these genes will have the desired effect? No, of course you do – and you don’t know what side effects – after every announcement a a new aging hypothesis that sounds the least bit plausible, there’s always the same bubble of enthusiasm. which dies away as the latest miracle drug or hormone or growth factor proves less than expected. It’s like Aubrey de Grey trying to re-engineer the cell he has only the slightest understanding of (like the rest of us) to somehow be ‘better’! As though we paltry humans even understand how a single eukaryotic cell works, much less ‘improve’ it – we do not, except a very little bit, and more and more I think a lot of what we think we know is wrong (I’ve seen many times). Read my paper, maybe you’ll understand.

  2. Thanks for these thoughts, Harold. You may be right that changing gene expression “by hand” is a dead end. But I think you might like the concept better if you think of it as a temporary way to get enough of the right hormones into the blood on an ongoing basis, over a period of months. If your hunch and mine is correct, then these hormones in the blood will re-program the epigenetics and the CRISPR handiwork can fade away.

    Of course, we won’t know until we try it. This is a plan I would like to see someone implement in a mouse sometime in the next 2 years.

  3. I don’t think it’s necessary, as Mr. Katcher writes, to have wide-ranging genomic control through CRISPR to attain useful therapies. What would be necessary is to determine what is the mechanism of varying secretion from the specific tissues and cells responsible for under- or over-expressed bloodborne factors.

    Is it expressed where it shouldn’t be? Silence it. Is it not expressed where it should be, full stop? Turn it on. If the amounts still aren’t correct, something else is responsible, and more sophisticated measures will need to be developed.

    I would wager that some progress and experimentation in this arena is far better than none.

      • True, trying to track the expression and epigenomic changes in the major genes related to (and including) just the single component we’re looking for doesn’t sound fun, especially compared to, say, just supplementing the thing we need or transplanting something that already successfully creates it.

        I was given the impression first reading about this technology that (used therapeutically) it might be most useful for existing, identified mutations, not so much for tight epigenetic control.

        But with better analytic techniques, perhaps we will see a future era in which personal tissue-specific epigenomics becomes available, and we use software to come up with a panel of therapies designed to target each and every differing gene in major secretory tissues to restore them as closely as possible to the same profiles the individual had in their youth (or that genetically similar people had in their youth).

        My point is, though, just because it is an exceedingly complicated task does not mean it is impossible or should not be speculated upon or researched.

    • Maybe. The price has dropped dramatically over the years, and is now affordable for (many) individuals. But what can we learn from comparing genomes? We can find genetic variations associated with longevity, for sure, but changing genes within a living organism is still the hard part. CRISPR may change that.
      -Josh

  4. Hi,

    I was just wondering if you have any evidence to back up the claim “The CRISPR process is much less efficient on heterochromatin compared to euchromatin”. Of course this makes sense, however where are the studies to show that this is the case and how CRISPR/CAS9 interacts with modified chromatin.

  5. Hello

    I have some basic question regrading expression of this system in plant cell.I am wondering that,how it work inside the cell.Like once we transferred our construct in plant cell with the help of T-Plasmid,then it integrate in to plant genome and expression of our clone will start.so my question rise here that
    1.) How RNA and CAS protein complex occur.Because i my thinking that translation of cas protein occur only in cytoplasm.How it goes again in nucleus and bind with gRNA and tracrRNA. So guide cas protein or they have any nucleus pore signal .

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