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
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
Edit DNA to find, insert, or remove any desired
sequence in any location.
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.
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
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.
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
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.
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.
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
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
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.
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
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.
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
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.
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.
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.
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
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 .
Columns Online: Electronic
reprints of about 180 "The Alternate View" columns by John G. Cramer,
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