Discovery Pola’s marshes. His advisor had found

Discovery of CRISPR

The story starts in the
Mediterranean port of Santa Pola on Spain’s Costa Blanca, where the

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beautiful coast and vast
salt marshes have for centuries attracted vacationers, flamingoes, and

commercial salt
producers. (The geography of the story is shown in Figure
2.) Francisco

Mojica, who grew up
nearby, frequented those beaches, and it was no surprise that, when he

began his doctoral
studies in 1989 at the University of Alicante, just up the coast, he joined a

laboratory working on Haloferax mediterranei, an archaeal
microbe with extreme salt tolerance

that had been isolated
from Santa Pola’s marshes. His advisor had found that the salt

concentration of the
growth medium appeared to affect the way in which restriction enzymes

cut the microbe’s
genome, and Mojica set out to characterize the altered fragments. In the first

DNA fragment he
examined, Mojica found a curious structure—multiple copies of a nearperfect,

roughly palindromic, repeated
sequence of 30 bases, separated by spacers of roughly

36 bases—that did not resemble any family
of repeats known in microbes (Mojica et al., 1993).

Figure 2. The
Twenty-Year Story of CRISPR Unfolded across Twelve Cities in Nine Countries

For each “chapter” in
the CRISPR “story,” the map shows the sites where the primary work occurred and

first submission dates
of the papers. Green circles refer to the early discovery of the CRISPR
system and its

function; red to the
genetic, molecular biological, and biochemical characterization; and blue to
the final step

of biological engineering to enable genome editing.

The 28-year-old graduate
student was captivated and devoted the next decade of his career to

unraveling the mystery.
He soon discovered similar repeats in the closely related H. volcanii, as

well as in more distant halophilic
archaea. Combing through the scientific literature, he also

spotted a connection
with eubacteria: a paper by a Japanese group (Ishino et al., 1987) had

mentioned a repeat
sequence in Escherichia coli
that had a similar structure, although no

sequence similarity, to
the Haloferax repeats.
These authors had made little of the observation,

but Mojica realized that
the presence of such similar structures in such distant microbes must

signal an important
function in prokaryotes. He wrote up a paper reporting this new
class of

repeats (Mojica
et al., 1995) before heading off for a short post-doctoral stint at Oxford.

Mojica returned home to
take up a faculty position at the University of Alicante. Because the

school had hardly any
start-up funds or lab space, he turned to bioinformatics to investigate the

strange repeats, which
he dubbed short regularly spaced repeats (SRSRs); the name wouldlater be
changed, at his suggestion, to clustered regularly interspaced palindromic

(CRISPR) (Jansen
et al., 2002; Mojica and Garrett, 2012).

By 2000, Mojica had
found CRISPR loci in 20 different microbes—including Mycobacterium

tuberculosis, Clostridium difficile, and the plague
bacteria Yersinia pestis (Mojica
et al., 2000).

Within 2 years,
researchers had doubled the census and cataloged key features of loci—

including the presence
of specific CRISPR-associated (cas) genes in the immediate vicinity,

which were presumably related to their
function (Jansen et al., 2002)

But what was the
function of the CRISPR system? Hypotheses abounded: it was variously

proposed to be involved
in gene regulation, replicon partitioning, DNA
repair, and other roles

and Garrett, 2012). But most of these guesses rested on little or no evidence,
and one

by one they proved to be
wrong. As with the discovery of CRISPR, the critical insight came

from bioinformatics.

CRISPR Is an Adaptive
Immune System

During the August
holiday in 2003, Mojica escaped the scorching heat of Santa Pola’s beaches

and took refuge in his
air-conditioned office in Alicante. By now the clear leader in the nascent

CRISPR field, he had
turned his focus from the repeats themselves to the spacers that

separated them. Using
his word processor, Mojica painstakingly extracted each spacer and

inserted it into the
BLAST program to search for similarity with any other known DNA

sequence. He had tried
this exercise before without success, but the DNA sequence databases

were continually
expanding and this time he struck gold. In a CRISPR locus that he had

recently sequenced
from an E. coli strain,
one of the spacers matched the sequence of a P1

phage that
infected many E. coli strains. However, the
particular strain carrying the spacer was

known to be resistant to
P1 infection. By the end of the week, he had slogged through 4,500

spacers. Of 88 spacers
with similarity to known sequences, two-thirds matched viruses or

conjugative plasmids
related to the microbe carrying the spacer. Mojica realized that

loci must encode the
instructions for an adaptive immune system that protected microbes

against specific

Mojica went out to
celebrate with colleagues over cognac and returned the next morning to

draft a paper. So began
an 18-month odyssey of frustration. Recognizing the importance of the

discovery, Mojica sent
the paper to Nature. In November 2003, the
journal rejected the paper

without seeking external
review; inexplicably, the editor claimed the key idea was already

known. In January 2004,
the Proceedings of the National Academy of
Sciences decided that

the paper lacked
sufficient “novelty and importance” to justify sending it out to review. Molecular

Microbiology and Nucleic Acid Research rejected the paper in
turn. By now desperate and

afraid of being scooped,
Mojica sent the paper to Journal of Molecular
Evolution. After 12

more months of review
and revision, the paper reporting CRISPR’s likely function finally

appeared on February 1,
2005 (Mojica et al., 2005).

At about the same time,
CRISPR was the focus of attention in another, rather unlikely, venue: a

unit of the French
Ministry of Defense, some 30 miles south of Paris. Gilles Vergnaud, a human

geneticist trained at
the Institut Pasteur, had received doctoral and post-doctoral support from

the Direction Générale
de l’Armement. When he completed his studies in 1987, he joined the

government agency to set
up its first molecular biology lab. For the next 10 years, Vergnaud

continued his work on
human genetics. But when intelligence reports in the late 1990s raised

concerns that Saddam
Hussein’s regime in Iraq was developing biological weapons, the Ministry of
Defense asked Vergnaud in 1997 to shift his group’s focus to forensic

—developing methods to
trace the source of pathogens based on subtle genetic differences

among strains.
Establishing a joint lab with the nearby Institute of Genetics and Microbiology

Université Paris-Sud, he
set out to use tandem-repeat polymorphisms—which were the

workhorse of forensic
DNA fingerprinting in humans—to characterize strains of the bacteria

responsible for anthrax
and plague.

The French Defense
Ministry had access to a unique trove of 61 Y.
pestis samples from a

plague outbreak in
Vietnam in 1964–1966. Vergnaud found that these closely related isolates

were identical at their
tandem-repeat loci—with a sole exception of a site that his colleague

Christine Pourcel
discovered was the CRISPR locus. The strains occasionally differed by the

presence of new spacers,
which were invariably acquired in a polarized fashion at the “front”

end of the CRISPR locus
(Pourcel et al., 2005). Strikingly, many of the new

corresponded to a prophage
present in the Y. pestis genome.
The authors proposed that the

CRISPR locus serves in a
defense mechanism—as they put it, poetically, “CRISPRs may

represent a memory of ‘past
genetic aggressions.'” Vergnaud’s efforts to publish their

met the same resistance
as Mojica’s. The paper was rejected from the Proceedings
of the

National Academy of Sciences,
Journal of Bacteriology, Nucleic
Acids Research, and Genome

Research, before being published in Microbiology on March 1, 2005.

Finally, a third
researcher—Alexander Bolotin, a Russian émigré who was a microbiologist at

the French National
Institute for Agricultural Research—also published a paper describing the

extrachromosomal origin
of CRISPR, in Microbiology in
September 2005 (Bolotin et al., 2005).

His report was actually
submitted a month after Mojica’s February 2005 paper had already

appeared—because his
submission to another journal had been rejected. Notably, Bolotin was

the first to speculate
how CRISPR conferred immunity—proposing that transcripts from the

CRISPR locus worked by anti-sense
RNA inhibition of phage gene expression. Although

reasonable, the guess
would prove to be wrong.