From Science, the flagship publication of the American Association for the Advancement of Science:
I speak biology fluently, but the molecular complexities of the novel
genome-editing tool called CRISPR left me as befuddled as when I peruse
descriptions of the inflationary universe. So I decided to test what
one investigator told me: CRISPR (for "clustered regularly interspaced
short palindromic repeats") may sound intimidating, but it is so simple
to use that "any idiot" could do it.
I would give it a try.
CRISPR works best at crippling, or knocking out, genes, so that's how
I choose to use it. But I aim high: I target an immune gene that, I
theorize, could lead to insights into reducing the harm done by Zika
virus. (My admittedly wild hypothesis is that the gene, CD32,
may help drive Zika virus to copy itself to higher levels if a person
was previously infected with dengue and has antibodies to that virus.)
Roland Wagner, a postdoc in the lab of Sumit Chanda at the
Sanford Burnham Prebys Medical Discovery Institute in San Diego,
California, agreed to serve as my CRISPR sensei. An experienced rock
climber originally from Austria, Wagner approaches everything
methodically. He pulls up the sequence of the CD32 gene, which
has five distinct protein-coding regions. If we cut the DNA in one
region, the gene most likely would be knocked out: It would no longer
make its protein.
CRISPR uses a guide made of RNA to direct molecular scissors—part of
the CRISPR-associated protein, or Cas9—to exact spots in a genome. We
could buy the guide RNA (gRNA), but the idea appalls Wagner. "I would
assume it's probably $500 to buy the gRNA, but I wouldn't know," he
says. "We're making our own and we're spending about $5."
The gRNA sequence must complement a stretch of 20 nucleotides on the segment of the CD32
gene we want to cut. But the same DNA sequence could recur elsewhere in
the genome, leading the molecular scissors to cut in the wrong place.
Such "off-target" effects can cause mayhem, and eliminating them is a
key goal of those honing their CRISPR skills. To make the match more
specific, Cas9 requires an additional sequence flanking the targeted 20
nucleotides: N-G-G, in which "N" can be any nucleotide. Where Cas9 finds
N-G-G immediately following the 20 nucleotides, it attaches to and
opens the double helix, allowing the gRNA to bind. Cas9 then cuts each
strand of the DNA.
To homebrew our gRNA, Wagner copies the sequence of the CD32 segment
we've identified and pastes it into a freely available database,
Optimized CRISPR Design, that looks for a matching set of 20 nucleotides
followed by N-G-G. There are 41 options within CD32. The database scans
the entire human genome to see whether there are identical matches
elsewhere—potential sites of off-target cuts. We select a sequence that
appears unique, and then he goes to another website and orders a stretch
of DNA—an oligonucleotide—with that sequence.
The oligo arrives, and I lose my modern pipetting virginity. I have
not worked in a lab since I was an undergraduate more than 30 years ago.
Back then, I learned a pipetting technique that probably was invented
by Louis Pasteur: I put a finger in my mouth and then sucked up a
chemical into a thin glass tube, capping it with my fingertip when I had
drawn up enough.
Now, at Wagner's lab bench, I face a rack of fancy plastic
gizmos that look like squirt guns but enable users to suck up precise
microliters of liquid with a push of a button. My task is to pipette the
oligo from one tiny test tube into another. The second tube holds a
plasmid, which is a circular piece of DNA that will act as a Trojan
horse. This plasmid, customized for CRISPR experiments, already holds
the gene for Cas9. It also contains a 60-nucleotide "hairpin" sequence
that ultimately will attach to the 20 nucleotides I add to make the full
gRNA....MORE
