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Genome Editing: The CRISPR Revolution

by John G. Cramer

Alternate View Column AV-179
Keywords: DNA, genome, editing, matching, sequence, cut, strand, helix
Published in the December-2015 issue of Analog Science Fiction & Fact Magazine;
This column was written and submitted 06/25/2015 and is copyrighted 2015 by John G. Cramer.
All rights reserved. No part may be reproduced in any form without
the explicit permission of the author.

    

This column is about CRISPR, a new technique for editing, deleting, and inserting DNA coding into any genome.  Synthetic molecular biology, in its present stage of development, has a useful toolbox of techniques for DNA manipulation.  These techniques can:

         Read DNA and RNA sequences, identifying the sequence of bases (T:C:A:G) for DNA and (U:C:A:G) for RNA;

         Write or synthesize DNA and RNA sequences;

         Amplify DNA exponentially using the polymerase chain reaction (PCR), a heat-cycling technique that doubles copies of DNA sequences with each cycle;

         Cut DNA sequences using restriction enzymes (about 3,000 known) that cleave at a 4- to 6-base site recognized by the particular enzyme; and

         Edit DNA to find, insert, or remove any desired sequence in any location.

Up to now, edits using zinc-finger or TAL-effector nuclease have been, difficult, unreliable, time-consuming, and expensive.  Nuclease techniques require custom sequencing of many bases to produce the needed nuclease sequence and need a separate nuclease sequence for each side of the DNA double helix.  Further, cutting the identified DNA requires long time delays.  Gene editing with such techniques is technically demanding, time consuming, not completely reliable, and costs tens of thousands of dollars per edit.

The new gene editing technique, CRISPR/Cas9, is a cheap and effective allternative.  CRISPR, (Clustered Regularly Interspaced Short Palindromic Repeats), refers to identical repeating base sequences discovered in the genome of some bacteria in 1986.  These sequences were found to alternate with many 20-base sections, later identified as a "rogues gallery" of DNA samples from viruses that attack bacteria.

It was discovered in 2011 that the Cas9 protein, found in the bacterium Streptococcus pyogenes, uses these sequences in an antiviral immune system.  When the CRISPER region is transcribed to messenger RNA, it creates virus-sample+tail "guide RNA" that programs Cas9 to target the matching region of virus DNA and to cut both of its DNA strands, thereby disabling the virus.

Coding DNA strand is read from its starting "5-prime" end to its concluding "3-prime" end.  The 5' end of guide RNA contains the sequence that will dock with a complementary sequence in the DNA of the target virus, and the 3' end finishes with the CRISPR sequence, a tail that is recognized by Cas9 and that docks with a separate "tracr RNA" sequence in a recess of the Cas9 structure.  With this programming in place, Cas9 moves along both strands of virus double-helix, locates the matching sequence on one of these, and cuts both DNA strands beyond the location of the match.  The virus, cut in two pieces, is immobilized, protecting the bacterium from viral invasion.   Even if the two cut halves subsequently rejoin, they should lose enough base-pairs to suffer a "frame-shift" mutation that prevents the viral DNA from performing its function.

CRISPR/Cas9 follows certain rules.  The target DNA sequence on the virus must have a length of exactly 20 base pairs.  This provides good target selectivity, because there is only 1 chance in 420 or 2.88 x 1017 that some random DNA elsewhere will match the target sequence.  Further, the target sequence must be immediately adjacent to a "PAM spacer", a sequence that varies with the bacterium from which the Cas9 was obtained.  The widely-used Cas9 enzyme from Streptococcus pyogenes recognizes the PAM sequence NGG, where N means any base.  The viral DNA in the CRISPR region of the parent bacterium never has this sequence, preventing Cas9 from attacking the "virus sample" DNA sequence stored in its own parent bacterium.

Researchers at Berkley have improved on Nature by joining the tracr RNA sequence to the 3' end of the guide RNA sequence, producing a chimera "hairpin+tail" shape that fits the Cas9 enzyme recess.  This is simpler, because no separate tracr RNA is needed.

Multiplexing tests of Cas9 have been performed in which 73 different guide RNA sequences were introduced at once, targeting 73 different DNA patterns with the same Cas9 enzyme.  These tests indicated no loss of effectiveness in finding and cutting all the targeted sequences in the same operation.

Thus, by synthesizing fairly short RNA sequences, Cas9 can be made to find and cut any specific 20-base region of any DNA helix, whether viral, bacterial, plant, or animal.  This has broad implications for genome editing.  Commercial firms are already selling small "plasmid" DNA loops that generate a user-specified CRISPR guide RNA sequence and also generate Cas9 itself and associated enzymes, all in a single package that can be PCR-amplified and used with cell cultures or test animals.  The CRISPR-Cas9 technique is simple to implement, and the cost per edit is only around $100.


How is CRISPR used?  The simplest application is "gene knockout of a selected gene in a plant or animal genome, often done to learn the gene's function.  If the sequence of the genome is known, one identifies a 20-base+ PAM-spacer region, synthesizes guide RNA that targets that region, and sends programmed Cas9 to cut it.  Most of the genes cut in this way will be inactivated, and researchers can observe the inactivation's effect on a studied cell or organism.

Alternatively, one can insert new genetic coding by supplying an additional "homologous" DNA strand that matches both cut ends of the break.  One can also do a "snip out and replace" by synthesizing two RNA sequences that program Cas9 to cut the DNA in two locations, thereby snipping out a complete unwanted gene sequence.  If one also supplies a replacement homologous DNA sequence that matches both cut ends, the created gap will be filled with a new sequence.  All of these procedures have been demonstrated in the laboratory, applied to human cells and to cells of yeast, plants, fruit flies, zebra fish, mice, rats, frogs, and monkeys, and have been shown to have a reasonably high success ratio.

There are also synthesized variants of the wild Cas9 enzyme that (1) cut only the targeted side of the DNA helix, (2) cut only the opposite side, or (3) cut neither side, but only find the target sequence and park there.  If the no-cut enzyme is coupled to an enzyme that activates or suppresses gene expression, the unit can switch on or off a targeted gene, initiating or suppressing the production of proteins.

Tests have shown that Cas8 sometimes cuts non-targeted sequences that only partially match the guide RNA pattern.  However, the probability of such off-target cuts can be greatly minimized by using two single-cut Cas9 enzymes that cut opposite sides of a DNA helix that are separately targeted.


The applications of the CRISPR/Cas9 technique are just beginning to emerge from laboratories around the world.  One can expect that among the first commercial applications will be the production of drug proteins by gene splicing.  This technique began in the 1970s with the production of insulin with recombinant DNA, using a restriction enzyme to cut a bacterial DNA loop and splice in a sequence that produced insulin.  With CRISPR, the production of similar drug proteins becomes much easier and faster, with greatly reduced development costs.

Another obvious CRISPR application is attacking any double-stranded DNA virus that had been sequenced.  Such a virus could be rapidly targeted by synthesizing a short RNA string that would command CAS9 to cut and immobilize it.  Further, recent work has shown that CAS9 can be modified to target and cut strands of viral RNA, so that retroviruses can also be attacked.

Genetic manipulation of plants is another application of CRISPR.  This, of course, has been going on for many years using restriction enzymes and other expensive and low-yield techniques.  It is estimated that 60% of the food in our grocery stores includes ingredients from genetically modified plants.  However, the advent of CRISPR should make the design of food crops easier, faster, and cheaper and lead to new crops that are more adaptable to marginal growing conditions and more resistant to disease and insect pests.  This will introduce needed competition into an area that presently requires heavy investment of time and money.  One can hope that this increased competition with the big producers of GMO monoculture crops, who have used armies of lawyers to threaten and sue farmers to the detriment of agriculture, will lead to more open research, better crops, and fewer lawsuits.

Genetic manipulation of animal genomes with CRISPER is already a growth area, because, using the gene-knockout technique described above, researchers can turn off portions of the genome, one gene at a time, and discover their functions.  Example: CRISPR-based "gene-drive" techniques may lead to malaria-resistant mosquitoes.


Finally we come to the question of applications of CRISPR to the human genome.  This area is an ethical minefield.  For example, there are many genetic diseases that presently have no known cure.  These can be divided into diseases missing a needed gene and diseases in which a mutated gene is causing problems and needs to be turned off.

The capability of CRISPR to insert or remove a given gene in a living organism has its limits in efficiency.  At present under ideal conditions, application of CRISPR might at best insert or knock out genes in 30-40% of an organism's cells.   To insert a good gene, this success fraction might be sufficient.  Bad gene knockout might require a higher success fraction, but even at the available level might at least reduce symptoms.  CRISPR is already being used to treat human genetic diseases, but how far should this go?

There are also rare beneficial genes, identified in only a few individuals, that would be good for all of us to have.  The gene MSTN leads to lean muscles, LRP5 to extra-strong bones, GHR and GH reduce cancer risk, PCSK9 reduces coronary disease, CCR5 and FUT2 gives virus resistance, SLC30A0 gives resistance to diabetes, and APP gives resistance to Alzheimer's.  Should we consider using CRISPR to insert these beneficial genes into the genomes of all humans?

Human aging is another potential CRISPR impact area.  The chromosomes of each cell have telomere-ends that grow shorter with each cell division, and when the telomeres are gone, the cell cannot divide, producing the symptoms of old age.  (See my Column #88 in the February-1998 Analog.)  The human genome contains the coding for the enzyme telomerase, switched off in adults, which systematically goes to chromosome ends and rebuilds the cell's telomeres.  There are also other switched-off protein production genes like WRN related to aging, and it would also be desirable to switch off production of harmful proteins like NFκB, TGF-β, and JAK/STAT.

With no-cut Cas9, one could produce guide RNA that would target switched-off genes and activate them (or vice-versa), restoring the telomeres of the cell and producing or suppressing other age-related proteins.  We do not yet know enough about the aging process to know if this would actually rejuvenate an individual, but it would be a good start (and I'd volunteer.)

And so, the ethical arguments go on.  God made the human genome, and we should not change it.  Gene insertion, deletion, or activation might have unforeseen consequences and unknown side effects.  Aging is a natural process, and we should not interfere with it.  If you allow modification of the human genome, where do you stop, and when would the results cease to be human?  And so on.

Some ethicists suggest the "Heinlein guideline", based of Robert A. Heinlein's 1948 novel Beyond This Horizon, in which humanity has used genetic selection from the natural variations of the parents' combined genomes to produce a human population with increased health, strength, longevity, and intelligence.   But the society has limited the process to selection only from human genes that are available, rather than performing editing, insertion, and deletion.  Is that where we should draw the line?  In China , researchers may have already stepped over it by using CRISPR on 86 non-viable human embryos, 28 of which were successfully spliced.  The NIH plans a 2015 summit meeting soon to decide where the lines are drawn.


References:

Readers are encouraged to search the Internet for "CRISPR" to find many articles and videos discussing the techniques described here.


John Cramer's new book:  a non-fiction work describing his Transactional Interpretation of quantum mechanics, The Quantum Handshake - Entanglement, Nonlocality, and Transactions, (Springer, January-2016) is available online as a printed or eBook at:  http://www.springer.com/gp/book/9783319246406 . 

SF Novels by John Cramer:  my two hard SF novels, Twistor and Einstein's Bridge, are newly released as eBooks by Book View Cafe and are available at : http://bookviewcafe.com/bookstore/?s=Cramer .

AV Columns Online: Electronic reprints of about 180 "The Alternate View" columns by John G. Cramer, previously published in Analog , are available online at: http://www.npl.washington.edu/av.


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