{"id":287,"date":"2014-09-15T21:32:47","date_gmt":"2014-09-15T21:32:47","guid":{"rendered":"http:\/\/joshmitteldorf.peachpuff-wolverine-566518.hostingersite.com\/?p=287"},"modified":"2016-06-25T10:52:06","modified_gmt":"2016-06-25T10:52:06","slug":"crispr-in-your-future","status":"publish","type":"post","link":"https:\/\/scienceblog.com\/joshmitteldorf\/2014\/09\/15\/crispr-in-your-future\/","title":{"rendered":"CRISPR in your Future"},"content":{"rendered":"

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. \u00a0Last year, they figured out how to insert and delete genes. \u00a0This year there are methods for repressing and perhaps promoting genes (epigenetically, without modifying the genome) using CRISPR-derived technology. \u00a0Enthusiasts say they will soon be able to turn genes on and off at will. \u00a0It is my belief (I\u2019m not alone<\/a>) that aging is controlled largely by epigenetics\u2014what genes are turned on, when, and where. \u00a0Rapid 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. \u00a0It 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. \u00a0How do you get the CRISPR protein\/RNA complex into the cell nucleus? \u00a0<\/em><\/p>\n

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –<\/p>\n

The first generation of genetic engineering was turned to therapeutic use by means of genetically-modified viruses. \u00a0Viruses 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. \u00a0For 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. \u00a0Second, the virus is denatured, crippled so that it has a limited lifetime in the host, and won\u2019t keep multiplying at the host\u2019s expense. \u00a0(The host is the patient.)<\/p>\n

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. \u00a0Adenoviruses 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. \u00a0Nevertheless, there are about 2,000 clinical trials currently approved world-wide.<\/p>\n

Zinc finger nucleases and TALEN are second-generation technology. \u00a0These 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. \u00a0Potentially, a gene can then be removed or inserted. \u00a0The principal disadvantages are that they are time-consuming and therefore expensive. \u00a0It is not easy to engineer a protein that reliably binds to a particular target stretch of DNA.<\/p>\n

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. \u00a0Viruses (bacteriophages) can infect bacteria and insert their own viral genes into the bacteria\u2019s genome. \u00a0CRISPR-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.<\/p>\n

This nifty defense evolved in bacteria and archaea, but not in animals or plants. \u00a0Now, 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.<\/p>\n

(The acronym stands for C<\/b>lustered R<\/b>egulary-I<\/b>nterspaced S<\/b>hort P<\/b>alindromic R<\/b>epeats. \u00a0What that means, and why there should be little palindromes spread through bacterial DNA are questions for another day, because they don\u2019t really help understand how CRISPR works, its potential and its limitations.)<\/p>\n

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. \u00a0The Cas9 splicing enzyme then finds the spot that matches the complement of the gRNA, and that\u2019s were it does its job. \u00a0Curiously, 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.<\/p>\n

\u00a0<\/strong><\/strong><\/p>\n

CRISPRi<\/b><\/p>\n

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. \u00a0(The \u201ci\u201d is for \u201cinterference\u201d and the acronym is intended to be reminiscent of RNAi, or RNA interference<\/a>, which is another second-generation technology, useful for silencing genes only.) \u00a0With CRISPRi, tags are attached to the DNA at a target location such that they interfere with transcription of a gene in progress. \u00a0Potentially, CRISPR can be adapted to promote genes as well, but this is more challenging<\/a>. \u00a0It is in the promise of full epigenetic control that the most exciting applications lie, in my opinion.<\/p>\n

\u00a0<\/strong><\/strong><\/p>\n

Delivery<\/b><\/p>\n

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. \u00a0It can be micro-injected into the cell nucleus, but this is practical only for experiments, one cell at a time. \u00a0CRISPR kits<\/a> are being sold as plasmids, which is their original progeny in bacteria. \u00a0Plasmids are small loops of DNA, commonly exchanged by bacteria, but foreign to animal and plant cells. \u00a0There are papers<\/a> describing adenovirus applications that combine with CRISPR to offer both control and penetration, and these are so far in early demonstration stages.<\/p>\n

\u00a0<\/strong><\/strong><\/p>\n

Active and Inactive DNA<\/b><\/p>\n

Sewing thread is made of multiple, tiny fibers twisted together. \u00a0The twisted structure has an integrity of its own, but it\u2019s liable to become tangled and knotted, so we keep it wound neatly on a spool until we need it. \u00a0The cell does the same thing with its DNA. \u00a0The twisted structure is the double helix. \u00a0And the DNA strand is so long that it\u2019s liable to become tangled. \u00a0(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.) \u00a0The spools are protein molecules called histones, and threads of DNA are wound around them for orderly storage. \u00a0Each chromosome is a continuous thread of DNA, and there are many spools along its length. \u00a0At 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<\/a>, and it is inactive, not available to be transcribed into proteins. \u00a0Unspooled DNA is euchromatin<\/a>, and this is the active form of DNA, ready to be transcribed.<\/p>\n

So what happens if a CRISPR unit (a Cas enzyme) comes along that is targeted to a part of the DNA that\u2019s tightly wound up as heterochromatin? \u00a0Not much happens. \u00a0The CRISPR process is much less efficient on heterchromatin compared to euchromatin. \u00a0Imagine a reader scanning through a book looking for a particular phrase. \u00a0The process is much more likely to work if the book is open. \u00a0This is another challenge for\u00a0realizing the potential of CRISPR.<\/p>\n

\u00a0<\/strong><\/strong><\/p>\n

Which genes to turn on and turn off?<\/b><\/p>\n

I wrote a series of blog posts on this question last year.<\/p>\n

Hormones that we lose as we age<\/a> include melatonin, thyroxine, DHEA and (recently announced) GDF11<\/p>\n

Hormones that are overexpressed<\/a>, and we need to repress or block include NF\u03baB, TGF-\u03b2\u00a0and (recently announced) JAK\/STAT signals<\/p>\n

\u00a0<\/strong><\/strong><\/p>\n

The Right Technology for Anti-Aging Remedies<\/b><\/p>\n

I\u2019m not ready to have my genes replaced, thank you very much. \u00a0I think that there are genes that are associated with longevity, and several together might add a decade or more to life expectancy. \u00a0But replacing genes is permanent, and it\u2019s based on a technology fraught with unexpected side-effects. \u00a0Besides, my body already knows how to be young. \u00a0When it was young, it had the same genes it had now, but the epigenetics\u2014the set of genes turned on and off was somewhat different. \u00a0I\u2019m willing to bet that restoring a young epigenetic state to my same old genes will make me young, and that\u2019s why I\u2019m pumped about the CRISPR technology.<\/p>\n

\u00a0<\/strong><\/strong><\/p>\n

Read more:<\/p>\n

Fore-Cas-t from The Scientist
\n<\/a>Kurzweil AI on CRISPR gene therapy
\n<\/a>Cas9 as a Versatile Tool for Engineering Biology
\n<\/a>Comparison of Zinc Finger, TALEN, and CRISPR
\n<\/a>Adenoviral vector delivery ofRNA-guided CRISPR
\n<\/a>CRISPR-Cas systems for editing, regulating and targeting genomes
\n<\/a>CRISPRi explained at a technical level
\n<\/a>On-line discussion of speculating about use of CRISPR as a gene promoter<\/a><\/p>\n","protected":false},"excerpt":{"rendered":"

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. \u00a0Last year, they figured out how to insert and delete genes. \u00a0This year there are methods for repressing and perhaps promoting genes (epigenetically, without modifying the genome) … Read more<\/a><\/p>\n","protected":false},"author":65,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_jetpack_newsletter_access":"","_jetpack_dont_email_post_to_subs":false,"_jetpack_newsletter_tier_id":0,"_jetpack_memberships_contains_paywalled_content":false,"_jetpack_memberships_contains_paid_content":false,"footnotes":"","jetpack_post_was_ever_published":false},"categories":[1],"tags":[],"class_list":["post-287","post","type-post","status-publish","format-standard","hentry","category-uncategorized"],"yoast_head":"\nCRISPR in your Future - Josh Mitteldorf<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/scienceblog.com\/joshmitteldorf\/2014\/09\/15\/crispr-in-your-future\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"CRISPR in your Future\" \/>\n<meta property=\"og:description\" content=\"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. \u00a0Last year, they figured out how to insert and delete genes. \u00a0This year there are methods for repressing and perhaps promoting genes (epigenetically, without modifying the genome) ... 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The surprising fact that our bodies are genetically programmed to age and to die offers an enormous opportunity for medical intervention. It may be that therapies to slow the progress of aging need not repair or regenerate anything, but only need to interfere with an existing program of self-destruction. Mitteldorf has taught a weekly yoga class for thirty years. He is an advocate for vigorous self care, including exercise, meditation and caloric restriction. After earning a PhD in astrophysicist, Mitteldorf moved to evolutionary biology as a primary field in 1996. He has taught at Harvard, Berkeley, Bryn Mawr, LaSalle and Temple University. He is presently affiliated with MIT as a visiting scholar. In private life, Mitteldorf is an advocate for election integrity as well as public health. He is an avid amateur musician, playing piano in chamber groups, French horn in community orchestras. His two daughters are among the first children adopted from China in the mid-1980s. Much to the surprise of evolutionary biologists, genetic experiments indicate that aging has been selected as an adaptation for its own sake. This poses a conundrum: the impact of aging on individual fitness is wholly negative, so aging must be regarded as a kind of evolutionary altruism. Unlike other forms of evolutionary altruism, aging offers benefits to the community that are weak, and not well focussed on near kin of the altruist. 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The surprising fact that our bodies are genetically programmed to age and to die offers an enormous opportunity for medical intervention. It may be that therapies to slow the progress of aging need not repair or regenerate anything, but only need to interfere with an existing program of self-destruction. Mitteldorf has taught a weekly yoga class for thirty years. He is an advocate for vigorous self care, including exercise, meditation and caloric restriction. After earning a PhD in astrophysicist, Mitteldorf moved to evolutionary biology as a primary field in 1996. He has taught at Harvard, Berkeley, Bryn Mawr, LaSalle and Temple University. He is presently affiliated with MIT as a visiting scholar. In private life, Mitteldorf is an advocate for election integrity as well as public health. He is an avid amateur musician, playing piano in chamber groups, French horn in community orchestras. His two daughters are among the first children adopted from China in the mid-1980s. Much to the surprise of evolutionary biologists, genetic experiments indicate that aging has been selected as an adaptation for its own sake. This poses a conundrum: the impact of aging on individual fitness is wholly negative, so aging must be regarded as a kind of evolutionary altruism. Unlike other forms of evolutionary altruism, aging offers benefits to the community that are weak, and not well focussed on near kin of the altruist. This makes the mechanism challenging to understand and to model. more at http:\/\/mathforum.org\/~josh","sameAs":["http:\/\/AgingAdvice.org"],"url":"https:\/\/scienceblog.com\/joshmitteldorf\/author\/joshmitteldorf\/"}]}},"jetpack_featured_media_url":"","jetpack_shortlink":"https:\/\/wp.me\/pgtN8h-4D","jetpack_sharing_enabled":true,"_links":{"self":[{"href":"https:\/\/scienceblog.com\/joshmitteldorf\/wp-json\/wp\/v2\/posts\/287","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/scienceblog.com\/joshmitteldorf\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/scienceblog.com\/joshmitteldorf\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/scienceblog.com\/joshmitteldorf\/wp-json\/wp\/v2\/users\/65"}],"replies":[{"embeddable":true,"href":"https:\/\/scienceblog.com\/joshmitteldorf\/wp-json\/wp\/v2\/comments?post=287"}],"version-history":[{"count":0,"href":"https:\/\/scienceblog.com\/joshmitteldorf\/wp-json\/wp\/v2\/posts\/287\/revisions"}],"wp:attachment":[{"href":"https:\/\/scienceblog.com\/joshmitteldorf\/wp-json\/wp\/v2\/media?parent=287"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/scienceblog.com\/joshmitteldorf\/wp-json\/wp\/v2\/categories?post=287"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/scienceblog.com\/joshmitteldorf\/wp-json\/wp\/v2\/tags?post=287"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}