REVIEW ARTICLE

PUBLISHED: 5 JUNE 2017 | VOLUME: 2 | ARTICLE NUMBER: 17092

A decade of discovery: CRISPR functions

and applications
Rodolphe Barrangou1* and Philippe Horvath2
This year marks the tenth anniversary of the identification of the biological function of CRISPR–Cas as adaptive immune systems
in bacteria. In just a decade, the characterization of CRISPR–Cas systems has established a novel means of adaptive immunity
in bacteria and archaea and deepened our understanding of the interplay between prokaryotes and their environment, and
CRISPR-based molecular machines have been repurposed to enable a genome editing revolution. Here, we look back on the
historical milestones that have paved the way for the discovery of CRISPR and its function, and discuss the related technologi-
cal applications that have emerged, with a focus on microbiology. Lastly, we provide a perspective on the impacts the field has
had on science and beyond.

T
he nearly ubiquitous presence of diverse microorganisms
throughout the planet is awe-inspiring, yet somewhat per-
plexing given the widespread occurrence of bacteriophages
(or phages) and viruses that infect them. Consequently, the constant
arms race between prokaryotes and viruses has yielded a powerful
and nimble arsenal of defence mechanisms, encompassing restric-
tion modification, toxin–antitoxin modules, abortive infection and
the recently characterized CRISPR–Cas system1. The latter provides
adaptive immunity in bacteria and archaea (Box 1), and hinges on
diverse and versatile molecular machines that have popularized
CRISPR not only as a compelling means to fend off viruses, but also
as a democratized technology broadly used in molecular biology.
Here, we examine the various eras of CRISPR history, and highlight
discoveries that have advanced the field. First, we discuss a series
of milestone discoveries that assembled the CRISPR puzzle, and
basic findings that unravelled the molecular mechanisms provid-
ing adaptive immunity in bacteria. Next, we provide an overview
of the technical developments that have advanced the CRISPR field
and generated the toolbox that enables a diverse set of historical and
contemporary applications. Lastly, we discuss how these seminal
discoveries and versatile technologies have impacted and are likely
to continue to shape the field of microbiology.
The humble beginnings and meteoric rise of CRISPR
The remarkable number of studies dedicated to clustered regularly
interspaced short palindromic repeats (CRISPR)2 and CRISPR-
associated sequences (Cas)3 published in recent years illustrates the
speed at which the field has evolved, and the scale at which CRISPR-
based technologies and applications have impacted the fields of
microbiology, biology and genetics4. Yet CRISPR remained surpris-
ingly mysterious and underappreciated for decades; indeed, in the
beginning, CRISPR was a genomic peculiarity that was mostly an
assembly inconvenience. Here, we discuss the evolution of the per-
ception of CRISPR and its significance by comparing the history of
CRISPR to familiar historical periods in human history, leading to
unexpected, yet compelling, modern times (Fig. 1).
Antiquity. It may come as a surprise to many that the CRISPR lit-
erature dates back to the late 1980s, when several groups repeat-
edly observed arrays of DNA repeats in the genomes of bacteria
and archaea. Indeed, years before the CRISPR acronym was actu-
ally coined, a number of microbial geneticists came across these
peculiar loci in the genomes of microorganisms, primarily focus-
ing on model organisms and pathogenic bacteria. Originally,
these series of interspaced and partially palindromic DNA repeats
were observed in an intergenic region upstream of the iap gene
in Escherichia coli 5,6. Similar arrays of repeated DNA were also
observed in human pathogens, such as Mycobacterium tuberculosis 7
and Streptococcus pyogenes 8, as well as genomes of two Haloferax
species9 and a filamentous cyanobacterium10. In these early days,
several acronyms such as DVR (direct variable repeats), TREP (tan-
dem repeats), LTRR (long tandemly repeated repetitive sequences),
SRSR (short regularly spaced repeats), LCTR (large clusters of tan-
dem repeats), and later SPIDR (spacer interspersed direct repeats)
were used by various authors to refer to loci containing short and
partially palindromic DNA tandem repeats interspaced by seem-
ingly random sequences. The advent of genome sequencing tech-
nologies in the 1990s was a tipping point providing a large set of
microbial genomes that carried these peculiar sequences, although
their origin and function remained unknown.
Middle ages. In the early 2000s, with increasing availability of
genome sequences for numerous and diverse bacteria and archaea,
two groups consistently and repeatedly observed the occurrence
of CRISPR arrays2,3,11, leading to the creation of the consensus
acronym ‘CRISPR’ in 2002 (ref. 3) (Fig.  1). Though the biological
function of those widespread genetic features remained enigmatic
for nearly 20 years after their initial discovery, several hypotheses
were originally provided, ranging from replicon partitioning 9,11 to
DNA repair 12.
The first clue regarding the biological function of CRISPR was
unearthed in 2005, when three distinct research groups nearly
concurrently made the observation that the seemingly random
sequences separating identical CRISPR repeats actually showed
homology to invasive DNA sequences, such as viruses and plas-
mids13–15 (Fig.  1). It was then postulated that CRISPR may con-
stitute a putative defence system in prokaryotes13,16 that might
function analogously to the eukaryotic RNA interference (RNAi)
system, even though there are no homologues of RNAi machinery
components among the Cas proteins16.

Department of Food, Bioprocessing, and Nutrition Sciences, North Carolina State University, 400 Dan Allen Drive, Campus Box 7624, Raleigh,
1

North Carolina 27695-7624, USA. 2DuPont Nutrition and Health, BP10, 86220 Dangé-Saint-Romain, France. *e-mail: rbarran@ncsu.edu

NATURE MICROBIOLOGY 2, 17092 (2017) | DOI: 10.1038/nmicrobiol.2017.92 | www.nature.com/naturemicrobiology 1

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REVIEW ARTICLE NATURE MICROBIOLOGY

Box 1 | CRISPR mechanism of action.

There are two classes of CRISPR–Cas systems based on the
nature of the effector nuclease that drives targeting: either a
multi-protein complex for class 1, or a single protein for class
2. In either case, cas operons are adjacent to the CRISPR array,
which is transcribed from the leader (L) promoter sequence.
Typically, cas1 and cas2 genes are present in most types and
subtypes across the two classes. For class 1 systems, a canoni-
cal type I CRISPR–Cas system is shown, with the signature Cas3
exonuclease and Cascade. For class 2 systems, a canonical type II
CRISPR–Cas system is shown, with the signature Cas9 endonu-
clease. First, during acquisition, a short viral DNA sequence is
incorporated as a novel spacer (blue rectangle) into the CRISPR
array, where each repeat is shown as a black diamond. This is
a Cas1–Cas2-dependent process, and novel spacers are inte-
grated in a polarized manner at the leader-end of the CRISPR
array. Secondly, during expression, the CRISPR array is tran-
scribed into pre-CRISPR RNA (pre-crRNA) and subsequently
processed into small, mature crRNAs that each contain a target-
ing portion derived from a unique CRISPR spacer. Thirdly, dur-
ing interference, crRNAs guide the Cas effector nuclease (yellow)
for sequence-specific targeting and cleavage of complementary
nucleic acid. Each type is defined by a dedicated Cas effector
nuclease (see inset panel). In class 1, type I systems, the Cas3 exo-
nuclease nicks and then chews the target DNA strand; in type III
systems, the Cas10 nuclease cleaves mRNAs in a ruler–anchor
mechanism manner, which is coupled in some systems to target
DNA degradation; targeting remains uncharacterized in type IV
systems. In class 2 systems, type II systems use the Cas9 endo-
nuclease to generate two nicks that yield a dsDNA break; type V
systems use Cas12 to generate two offset nicks; type VI systems
use Cas13 to generate a cut in the target mRNA and then pro-
cesses it non-specifically to yield collateral damage. For type I, II,
V and VI, interference relies on PAM sequences (star symbols) to
initiate target recognition.

Phage

Cas1 Cas2

Class 1 New repeat New spacer

cas3 cas1 cas2
cascade

Class 2 L L
cas9 cas1 cas2 Acquisition
CRISPR array Expression
Genes encoding Cas proteins

pre-crRNA

Type I Type III Type IV

Class 1
Cas3 Cas10 Csf1
crRNAs

Type II Type V Type VI

Class 2 Interference
Cas9 Cas12 Cas13

Cas

Modern period. Following on from the proposal of CRISPR as an
immune system in prokaryotes came the first demonstration of its
biological function. We had the privilege to be part of the team (at
Danisco, a Danish food ingredient company now owned by DuPont)
for which the various pieces required to solve the ‘CRISPR puzzle’
were readily available: in silico datasets containing the genomes of
bacteria encoding diverse CRISPR–Cas systems and the genomes of
bacteriophages able to infect them; the biological material, includ-
ing bacterial isolates that carried active CRISPR–Cas systems and
lytic phages that could readily infect these hosts; and enough clues
from the existing literature.
This work started in the early 2000s during attempts to
develop molecular methods for strain differentiation of the yogurt
bacterium Streptococcus thermophilus. We discovered the exist-
ence of CRISPR—still called SPIDR at that time—in this spe-
cies through a poster presenting the genome sequencing of strain
CNRZ1066 (ref. 17). Concurrently, we repeatedly came across
CRISPR loci while assembling the draft genomes of lactic acid bac-
teria commonly used in food manufacturing, including the pro-
biotic Lactobacillus acidophilus 18. More interestingly, in late 2002,
we had access to the draft genome sequence for another strain of
S.  thermophilus, LMD-9 (refs 17 and 19), an industrially relevant
strain provided by Danisco, for which virulent phages were avail-
able. We initially used CRISPR sequences to compare and contrast
numerous S. thermophilus strains from the Danisco culture collec-
tion and observed the conservation of some spacers across groups

2 NATURE MICROBIOLOGY 2, 17092 (2017) | DOI: 10.1038/nmicrobiol.2017.92 | www.nature.com/naturemicrobiology

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NATURE MICROBIOLOGY REVIEW ARTICLE
of strains. Remarkably, strains clustered in groups for which the
CRISPR genotypes correlated with phage resistance phenotypes.
Furthermore, we noticed that strains in our collection that had been Discovery of clustered repeats in E. coli5
isolated at various points in time (for instance, after phage chal-
lenges) did occasionally carry additional repeat–spacer sequences, Specific study of these clustered repeats in enterobacteria6

1990
suggesting phage-induced and time-dependent variability of
CRISPR sequences. Critically, we were able to sequence CRISPR loci
of industrial strains and their phage-resistant variants that had been
used and generated in the 1980s and 1990s, and then retroactively
shed light on the genomic basis for their phage resistance pheno-

Antiquity
type. By cross-referencing these data with phage genome sequences,
the link between the CRISPR genotype and the phage resistance Use of CRISPR for M. tuberculosis typing7
phenotype became noticeable, extending prior observations about

1995
an inverse correlation between the size of CRISPR loci and phage
Identification of CRISPRs in Haloferax9
propagation proportion in this species15. Next, we embarked on a
series of experiments using bacterial cultures and their lytic phages Description of CRISPRs in cyanobacteria10
to test the hypothesis that spacers get acquired during the genesis of
phage resistance. We were able to show that: (1) CRISPR loci have
the ability to uptake phage DNA as novel spacers during the adapta-
tion stage (Box 1); (2) altering the CRISPR spacer content is directly Use of CRISPR for S. pyogenes typing8
responsible for the gain, loss or exchange of phage resistance via

2000
addition, deletion or transplantation of CRISPR spacers, respec- Widespread occurrence of CRISPRs in bacteria and archaea11
tively; (3) cas genes are implicated in both the adaptation stage (csn2-
dependent integration of new spacers) and interference process DNA repair hypothesis for cas genes12
CRISPR name coined; description of cas genes2,3
Middle ages
(cas9-dependent resistance to lytic phages) (Box  1). These results
were published in the seminal 2007 study establishing CRISPR–Cas
as the adaptive immune system of bacteria20, essentially launching
the CRISPR field ‘renaissance’ (Fig. 1).
2005

While this may seem evident in hindsight, testing the hypoth- Spacers probably derive from foreign genetic elements13,14,15
esis that CRISPR–Cas systems are involved in phage resistance in
bacteria was not straightforward, given the need to carry out these Putative RNAi-based mechanism proposed16
experiments in a genetically challenging species, S. thermophilus, for CRISPR–Cas is an adaptive immune system against viruses
in S. thermophilus20
which molecular biology reagents and genome alteration tools were
Discovery of the proto-spacer adjacent motif in S. thermophilus31,32
somewhat limited at the time. Nevertheless, the S.  thermophilus
Modern period

Immunity is mediated by small CRISPR RNAs in E. coli28

model21 was surprisingly the first CRISPR–Cas system in which
Plasmid dsDNA is the target in S. epidermidis29
adaptation20, biogenesis of CRISPR RNAs (crRNAs)22,23 and inter- ssRNA is the target in P. furiosus38
2010

ference24 were characterized in native conditions; recently, these

Precise cleavage of target DNA by Cas9 in S. thermophilus24
steps were also characterized in Pseudomonas aeruginosa25–27. Discovery of the tracrRNA in S. pyogenes36
Within a year, two additional important discoveries were made: Heterologous transfer of a complete CRISPR–Cas system37
establishing that CRISPR arrays are transcribed and processed
Reprogrammable, Cas9-based DNA cleavage44,45
into crRNAs that guide Cas effector proteins, such as Cascade Cas9-based genome editing of human cells46,47 and bacteria48
(CRISPR-associated complex for antiviral defense)28; and that DNA
Contemporary period

is the target of CRISPR-encoded immunity, with this system being Cas9 structure109,111
Cas9-based eradication of latent HIV infection82
able to provide a barrier to plasmid transfer between bacteria29
2015

(Fig. 1; Box 1).
Having established CRISPR as a DNA-encoded, RNA-mediated
and DNA-targeting immune system, a series of subsequent studies
delved into the biochemical and genetic processes underpinning the
mechanism of action30 (Box 1). One critical example is the discovery
of the occurrence and conservation of a CRISPR motif flanking the
targeted phage sequences31–33, which was renamed the protospacer
adjacent motif (PAM)33. Furthermore, it was demonstrated that
Cas9 is an endonuclease that precisely generates blunt DNA cleavage
exactly 3 nucleotides from the 3ʹ edge of the protospacer sequence24.
Cas9-mediated cleavage was also shown to be mediated by a seed
sequence, driving the specificity of targeting and intimacy of the
guide RNA:target DNA duplex 34,35. Subsequently in 2011, it was
shown that in type II systems, an ancillary RNA, the trans-encoded
crRNA (tracrRNA), is necessary to create a dual RNA guide for Cas9
(ref. 36). This was also the year when it was first established that
CRISPR–Cas systems can be transferred to distant bacterial species,
and reprogrammed to target plasmids and phages37 (Fig. 1).
In light of these studies, it became increasingly clear that there
are numerous and diverse CRISPR–Cas systems in nature that share
commonalities (DNA-encoded, RNA-mediated, nucleic acid target-
ing), but also carry out idiosyncrasies (various guide RNA types and
Cpf1 is a single RNA-guided nuclease134
Discovery of anti-CRISPR proteins against Cas9 in N. meningitidis65
Figure 1 | CRISPR milestones and seminal discoveries. Select publications
across the four listed eras are shown on the right. S. epidermidis,
Staphylococcus epidermidis; P. furiosus, Pyrococcus furiosus; N. meningitidis,
Neisseria meningitidis.
structures, as well as cleavage outcomes), especially with regard to
the type of nucleic acid targeted as some CRISPR–Cas systems can
actually target RNA38,39. Currently, CRISPR–Cas systems are cat-
egorized into two main classes: class 1 has a multi-protein effector
complex, whereas class 2 has a single effector protein. The classifi-
cation also includes 6 main types (I–VI) and over 19 subtypes40–43,
based on the CRISPR and Cas machinery and the mode of action
involved (Box 2).
The ‘modern period’ culminates with two milestone studies pub-
lished in the summer of 2012 showing that Cas9 is an endonuclease

NATURE MICROBIOLOGY 2, 17092 (2017) | DOI: 10.1038/nmicrobiol.2017.92 | www.nature.com/naturemicrobiology 3

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REVIEW ARTICLE NATURE MICROBIOLOGY

Box 2 | Origin and evolution of CRISPR–Cas systems.

Bacterial and archaeal genomes comprise an extraordinarily
diverse assortment of CRISPR–Cas systems that have been classi-
fied into 2 main classes, 6 types and 19 subtypes42,142. In addition to
the hyper-variable nature of CRISPR arrays, which is largely due to
the ability to capture novel spacers during the adaptation stage, the
CRISPR-associated cas gene pool also displays an extreme diver-
sification, probably resulting from numerous rearrangements and
horizontal transfer events over time40,42. Despite this high level of
genetic complexity, most cas genes are organized into two main
modules that are respectively involved in adaptation and interfer-
ence functions, and their detailed comparative analysis has led
to a simple and plausible scenario for the origin and evolution
of the Cas machinery, in which mobile elements played a pivotal
role143–146. Indeed, cas1 homologues not associated with CRISPR–
Cas loci revealed a new prokaryotic superfamily of self-synthe-
sizing transposable elements (transposons) named casposons
that employ the Cas1-like endonuclease as a recombinase, via an
integration/excision mechanism similar to that responsible for
the integration of new spacers into CRISPR loci143–145,147. Such a
casposon, whose terminal inverted repeats might have evolved
into CRISPR repeats, would have provided the adaptation mod-
ule of a CRISPR–Cas system ancestor in thermophilic Archaea
after fusion with a cas10-like nuclease gene originally involved in
innate immunity, and other ancillary genes40,146. Further genetic
divergence and rearrangements probably gave rise to a diversity
of class 1 systems, which then spread horizontally within prokary-
otic genomes. Subsequently, a distinct group of non-autonomous
mobile elements named ISC (insertion sequences cas9-like, deriv-
ing from IS605 family transposons) potentially provided, by
recombination happening at multiple and independent instances
during evolution, the RuvC, HNH and/or HEPN endo(ribo)nucle-
ase domains of the various class 2 CRISPR–Cas systems135,146,148.

that uses RuvC and HNH motifs to generate dual nicks in target
DNA44, which can be reprogrammed using a single-guide RNA
(sgRNA), mimicking the dual crRNA:tracrRNA complex 45 (Fig. 1).
These discoveries arguably set the stage for the repurposing of
CRISPR-derived molecular scalpels.
Contemporary period. Within months, a number of laborato-
ries showed nearly concurrently that CRISPR–Cas9 molecular
machines can be repurposed to generate double-stranded breaks
in DNA and lead to genome editing using DNA repair systems46–48
(Fig. 1), giving rise to what has been termed the ‘CRISPR craze’49
and the democratization of genome editing 50,51.
Disruptive CRISPR-based technologies have since taken the
world by storm, based on a number of Cas-based molecular
machines (that is, Cas9, Cpf1, dCas9 (deactivated variants of Cas9))
that enable genome editing, transcriptional control52–54 and epige-
netic alterations55. The many advantages and features of Cas pro-
teins (programmability, transferability, affordability, specificity and
efficiency, amongst many) have empowered geneticists throughout
the world and the phylogenetic spectrum to alter at will the genome
and transcriptome of all genetically characterized species tested
to date.
Ironically, though CRISPR has been broadly adopted as a
genome editing technology, there are no clustered regularly inter-
spaced short palindromic repeats per  se in the tools used to alter
genetic sequences, but rather sgRNAs that direct Cas9 (or other)
nucleases for sequence-specific targeting of DNA. Notably, whereas
CRISPR is commonly perceived as a genome editor, Cas9 merely
cuts DNA (as initially intended when targeting phage DNA in bac-
teria), while the actual editing is generated by the endogenous DNA
repair pathways.
Current studies of CRISPR–Cas systems investigate the various
aspects of the mechanism of action, with foci on novel immunity
acquisition genetics and machinery 56–60, as well as elucidating the
molecular and structural basis of interference by Cas nucleases61–63,
and its evolutionary implications, notably with regards to host–
virus interactions25,64 and the impact of CRISPR immunity and
targeting countermeasures27,65–68.
CRISPR applications in microbiology
Traversing the valley of death in translational science equates to
bridging the gap between seminal basic research discoveries and
their broad impactful applications and implementation. Often,
success depends on the genesis of tools and technologies that ena-
ble users to perform valuable tasks with ease, speed and limited
resources. With a sense of déjà vu related to the discovery of restric-
tion enzymes from restriction-modification systems69, bacterial
phage defence systems also ushered a new set of tools for molecular
biology based on Cas nucleases, and a new array of CRISPR-based
applications for microbiologists. Indeed, the popularity of CRISPR
hinges on the portability of Cas nucleases, the programmability
of RNA guides, and the speed and efficiency with which CRISPR-
based machines can be manipulated.
The many applications of CRISPR in eukaryotic organisms have
been extensively reviewed. For microbiologists, it is important to
note that both native and engineered systems can be repurposed or
exploited broadly in bacteria and archaea.
CRISPR-based genetic signatures. By essence, the CRISPR–Cas
adaptive immune system drives vaccination against invasive genetic
elements over time by capturing pieces of foreign DNA and insert-
ing them as novel spacers into the CRISPR array, iteratively build-
ing up a genetic record of immunization events20,31,70. These genetic
‘mug shots’ constitute a unique vaccination card that is hyper-vari-
able even amongst co-existing bacterial clones exposed to the same
lytic virus71. Thus, they constitute individualized immunization
records that serve as unique molecular signatures, and can be use-
ful to genotype bacteria and track their presence and occurrence.
Notably, due to the polarized mechanism of spacer acquisition at
the leader end of the CRISPR locus, leader-distal spacers provide
phylogenetic insights into ancestral origins of bacterial isolates,
while leader-proximal spacers provide insights into recent environ-
mental conditions of a bacterium (Fig. 2a). These loci thus consti-
tute valuable genetic and epidemiological markers for the tracking
of bacterial pathogens that are commonly problematic in the food
supply and responsible for infectious disease72,73, and analysis of
these CRISPR-based molecular signatures could be explored by
regulatory and monitoring agencies. Furthermore, unique com-
binations of spacers provide distinctive DNA fingerprints that
enable natural genetic tagging and molecular tracking of bacterial
isolates, which have been explored in industry and basic research.
For example, it was recently shown that adaptation can be repur-
posed to carry out molecular recording at a scalable level74 by prim-
ing CRISPR–Cas systems to acquire ‘donor’ DNA fragments as new
spacers into CRISPR arrays, yielding a unique molecular signature.
As our understanding and knowledge of microbiomes advance, it is
also possible to use hyper-variable CRISPR loci for high-resolution
genotyping of microbial populations64,75–77, with the ability to truly
assess the genetic diversity of a given species78. Early efforts to use
CRISPR to unravel genetic diversity in microbiomes have set the

4 NATURE MICROBIOLOGY 2, 17092 (2017) | DOI: 10.1038/nmicrobiol.2017.92 | www.nature.com/naturemicrobiology

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NATURE MICROBIOLOGY REVIEW ARTICLE
a Sequence-based genotyping b Anti-infective systems
Unique to Shared across Ancestral Phage
one cluster two clusters spacer

L
L Cas9
Identical spacers Cluster 1
L Target
DNA
L crRNA
L
Iterative additions Cluster 2
L
L
cas9
L
Cluster 3 Genes encoding Cas proteins CRISPR array
L
Unique profiles Cluster 4
L
Cluster 5

c CRISPR-based antimicrobials d Genome editing

CRISPR CRISPR
Phage L Phage L
DNA DNA cas9 or dcas9

crRNA crRNA
Cas3 Cas3 Cas9

DNA repair
dCas9
cas3
Gene silencing
Genes encoding Cas proteins

Figure 2 | CRISPR-based applications. a, Exploitation of CRISPR sequences for sequence-based genotyping in bacteria. Unique combinations of spacer
sequences enable unravelling of phylogenetic relationships between strains, with conserved ancestral spacers at the leader-distal end (which can
be shared across genetic clusters), and polymorphic content at the leader-proximal end (which can distinguish strains even within a closely related
cluster). b, CRISPR as anti-infective systems. Native CRISPR–Cas systems on the bacterial chromosome enable DNA-encoded, RNA-mediated (crRNA),
DNA-targeting cleavage of invasive viral DNA by the endonuclease Cas9, providing adaptive immunity. c, CRISPR-based antimicrobials. Endogenous
CRISPR–Cas systems can be repurposed by delivering, via engineered phages, self-targeting spacers that direct the Cas nucleases for chromosome
targeting and degradation, leading to lethal DNA damage generated by the native Cas3 exonuclease. d, Microbial genome editing. Viral delivery of a
Cas9 nuclease and a guide RNA can generate a dsDNA break at the target chromosomal location, which can be edited using the endogenous DNA
repair pathway(s). Alternatively, a deactivated version of Cas9 (dCas9) that can still bind to target sequences but does not cleave DNA enables
sequence-specific binding of dCas9 to block transcription, repressing target gene expression.

stage and developed the necessary tools to dig into complex bacte-
rial consortia and explore both bacterial composition and the inter-
play between hosts and viruses75,76,79,80.
A natural antiviral and anti-infective system. The basic biological
function of CRISPR–Cas immune systems can be used either natu-
rally or by engineering to build-up resistance against foreign mobile
genetic elements (such as bacteriophages, plasmids or transposons),
as well as to create a barrier against the uptake and dissemination
of undesirable genetic elements24 (such as antibiotic resistance
markers or toxin-encoding genes) (Fig. 2b). For example, iterative
vaccination events can be carried out to build-up broad-spectrum
phage resistance in industrial bacterial strains, and such strategies
have been commercialized on a global scale for dairy cultures70,81.
Additionally, there are also applications in generating beneficial
bacteria that promote human, animal and plant health (see below).
Moreover, these powerful antivirals have also been repurposed
to target viruses in eukaryotes, with noteworthy potential against
HIV82 and possibly all viruses that include a double-stranded DNA
(dsDNA) stage in their life cycle, as well as viruses that may present
a health risk during xenotransplantations83.
One originally unintended but practically desirable outcome of
effective phage sequence targeting by CRISPR is the selection of
escape phages31,84,85 through phage genome editing, essentially turn-
ing an escape mutation into an editing event by screening for muta-
tions of the target sequence86,87. Practically, as phage genome editing
tools are relatively primitive, targeting a particular viral sequence
using CRISPR can enable the user to screen and select for particu-
lar mutations in the PAM or protospacer sequence that enable the
phage to escape CRISPR targeting, essentially equating to ‘natural’
genome editing.
CRISPR-based antimicrobials. Whereas CRISPR–Cas systems
originally evolved to protect the integrity of bacterial genomes,
these systems can be repurposed for lethal self-targeting, culmi-
nating in bacterial elimination. Due to their lifestyle (single cells
with short lifespans and a high propensity and tolerance for genetic
fluidity) and limited access to complex and advanced DNA repair

NATURE MICROBIOLOGY 2, 17092 (2017) | DOI: 10.1038/nmicrobiol.2017.92 | www.nature.com/naturemicrobiology 5

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pathways (compared to their eukaryotic counterparts), bacteria are
particularly susceptible to genomic DNA damage. Consequently,
the primary outcome of CRISPR-encoded self-targeting in bacteria
is actually death88,89, rather than genome editing. This can be readily
harnessed to drive programmable and sequence-specific bacterial
cell death in microbial populations, especially when using the potent
and damaging type I CRISPR–Cas systems90 (Fig. 2c). Recently, sev-
eral studies have documented the selective antimicrobial potential
of CRISPR-based antimicrobials91,92, and opened new avenues for
the use of CRISPR-mediated self-targeting for the selective removal
of bacterial genotypes of interest (and the modulation of the com-
position of microbiomes) in preventing infectious disease, and also
reformulating bacterial consortia to promote the health of animals
and plants91. Strategically selecting the most damaging CRISPR–Cas
systems will promote lethality, and the naturally dominant type I
system, with the signature exonuclease Cas3, holds much potential
in the genesis of extensive DNA damage in bacteria93–98. Indeed,
generating more extensive DNA damage would be more challenging
to repair, or costlier to forego, consequently enabling more potent
eradication of a bacterial population. Furthermore, the widespread
nature of type I CRISPR–Cas systems99, notably in pathogenic bac-
teria such as Salmonella, E.  coli or Clostridium difficile, opens the
option to repurpose endogenous CRISPR effector nucleases to
selectively eliminate these pathogens.
Next-generation genome editing. As a nod to the repurposing of
microbial systems for genome editing in eukaryotes, microbiologists
have recently repurposed the sgRNA:Cas9 technology for genome
editing of bacteria (Fig.  2d). Indeed, the various genome editing
applications pioneered in human and animal cells have recently
been transferred (back) to bacteria to carry out genome editing
and transcriptional control, as well as genome-wide screens100.
Self-targeting can be used to alter the genetic content of bacte-
ria quickly and scalably, with exquisite specificity (since bacterial
genomes are so much smaller than their eukaryotic counterparts,
with much less repeated DNA) and efficiency (since prokaryotic
molecular machines work so well in bacteria). Furthermore, dCas9,
in which the RuvC and HNH nickase domains have been mutated,
have the ability to alter transcription patterns by either blocking
RNA polymerase (CRISPR interference, CRISPRi) or by triggering
gene expression (CRISPR activation, CRISPRa), enabling rerout-
ing of transcriptional pathways52–54,101. Again, microbiologists may
either exploit engineered, or repurpose endogenous, CRISPR–Cas
systems to drive transcriptional control102,103. While the popularity
and success of the CRISPR-fueled genome-editing revolution hinge
on the appetite built by RNAi and first-generation (ZFN-, TALEN-
and meganuclease-based) technologies104, the microbial genome
editing movement will rely on the adaptation of eukaryote-focused
tools and the continued progress of microbiome-focused research,
and identification of both the detrimental pathogens that need to
be eradicated, and the beneficial microorganisms that need to be
harnessed and improved.
Technological enhancements driven by an expanded exploita-
tion of diverse CRISPR–Cas systems105–107, as well as the develop-
ment of engineered variants with improved functionalities based on
structural insights108–113, set the stage for ever-increasing expansion
of the CRISPR–Cas toolbox. For example, rational engineering has
been implemented to alter PAM recognition and optimize efficiency
and specificity of Cas9 nucleases114–117. More recently, several studies
have focused on determining the structures of other class 2 effector
nucleases, including C2c1 and C2c2 (Cas13), unravelling the tar-
get recognition and cleavage mechanisms of action for both RNA-
guided DNA and RNA targeting, respectively 118–123. These studies
may open new avenues to use Cas-based nucleases for RNA target-
ing and detection, and enable a deeper understanding of the origin
and diversification of CRISPR–Cas systems (Box  2). Remarkably,
these reports highlight the interplay of homology and convergence
in the evolution of these effector nucleases, and illustrate how
nucleic acid targeting domains and catalytic sites have evolved and
specialized to effectively target various types of invasive molecules
in a nimble and diversified manner, perhaps reflecting the diversity
of invaders and predators. Importantly, the genesis of orthogonal
systems will enable the concurrent use and multiplexing of various
applications, such as metabolic rerouting by simultaneous upregula-
tion of specific pathways and downregulation of others124–126.
The ability to repurpose native systems in bacteria also enables
the screening of some rare natural variations, such as the excision
of expandable genomic islands127. Furthermore, CRISPR can be
readily used in combination with single-strand DNA recombineer-
ing (recombination-mediated genetic engineering) technologies to
quickly and efficiently modify the genomic content of industrial
workhorses and model microorganisms, such as lactobacilli, that
are widely used as commercial probiotics128. These technologies
have the potential to be implemented on a broad scale in all bacte-
ria of industrial interest, encompassing biomanufacturing for food,
agricultural and biotechnological applications129,130.
The CRISPR future is now
The current pace and scale at which CRISPR-based technologies are
being used is undeniably remarkable and is broadly perceived as the
tip of the iceberg, with unabating momentum in terms of science
and frenetic business interest. Indeed, from a scientific standpoint, it
appears our imagination is the sole limiting factor for what CRISPR-
based technologies can do, in terms of both applications and imple-
mentable species. The literature and citation quantitative patterns
are compelling and reflect rapid and nearly ubiquitous adoption
of this technology by most investigators studying genetics4,49.  The
accolades for various CRISPR pioneers have been humbling, though
arguably biased, and not always representative of the humble and
diverse bacterial roots of these fantastic molecular machines131,132.
From a business standpoint, the translational potential of this tech-
nology for both gene therapies and agricultural breeding have gen-
erated sizeable investments and impressive strategic partnerships
in fields that span the pharmaceutical, agricultural, food and bio-
technology industries. Of course, this enthusiasm is accompanied
by commensurately high expectations that may prove difficult to
live up to. Unfortunately, this has occasionally come at the cost of
controversial coverage, such as journalistic sensationalism and an
epic intellectual property battle, but this reflects the appetite for,
and potential of, this technology. Furthermore, this also creates a
tangible need to safely and responsibly manage the development
and implementation of CRISPR-based technologies with immedi-
ate implications for regulatory processes, ethical considerations,
and public relations133, which is sensitive in light of the stakes, per-
ception and previous shortcomings of GMO (genetically modified
organism)-associated technologies.
In many ways, the CRISPR story illustrates how science often
works, with a fitting reminder that the basic microbiological pro-
cesses driving the survival of bacteria often give rise to valuable
molecular tools, enzymes and technologies, together with the his-
torical involvement of a plethora of unsung heroes whom selflessly
advance science for the benefits of humankind. There is also a sense
of déjà vu with the serendipitous timing and assembly of dispersed
clues via a community-based scientific process with sometimes
parallel efforts and convergent foci. The collaborative, diverse and
multidisciplinary nature of the early CRISPR community built—
a decade ago—the strong momentum that enabled the progress
observed ever since.
The recent past illustrates the pace at which the field is advanc-
ing and the CRISPR literature exploding. Several recent studies
have reminded us that the bacterial dark matter still holds intrigu-
ing novel systems that can be repurposed for various applications,

6 NATURE MICROBIOLOGY 2, 17092 (2017) | DOI: 10.1038/nmicrobiol.2017.92 | www.nature.com/naturemicrobiology

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NATURE MICROBIOLOGY
such as a Cpf1 (Cas12) (ref. 134) and C2c1-2 (Cas13) (ref. 135), as
well as recently unearthed distant homologues of Cas9 and Cas12,
namely CasX and CasY (ref. 136). These molecular machines con-
tinue to diversify the target nucleic acid and cleavage types, as well
as the targetable landscape of sequences using PAM diversity 137.
Additionally, novel effectors, such as the anti-CRISPR proteins, can
be harnessed to control the activity of Cas enzymes either in nature
to evade CRISPR immunity 27, or in vitro to control the activity of
CRISPR-based technologies65. As always, many outstanding ques-
tions still remain, notably the presence of uncharacterized CRISPR–
Cas immune systems in nature and the rationale for their absence in
more than half of known bacterial species, as well as their biological
roles beyond adaptive immunity. Actually, in addition to providing
adaptive immunity, CRISPR systems have also been implicated in
biofilm formation and transcriptional control in bacteria, and there
may be additional functional roles138–141.
Certainly, this past decade has been filled with milestone
discoveries unravelling the genetic and biochemical basis of
CRISPR-based immunity in bacteria, as well as the repurposing
of native and engineered systems for a plethora of applications,
and we already look forward to a promising upcoming decade of
additional advances.
Received 23 December 2016; accepted 5 May 2017;
published 5 June 2017
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Acknowledgements
R.B. is supported by funds from North Carolina State University and the North Carolina
Ag. Foundation. The authors would like to thank their colleagues and collaborators for
their contributions and insights, and for having the privilege to share the CRISPR journey.
Author contributions
R.B. and P.H. wrote the manuscript.
Additional information
Reprints and permissions information is available at www.nature.com/reprints.
Correspondence should be addressed to R.B.
How to cite this article: Barrangou, R. & Horvath, P. A decade of discovery: CRISPR
functions and applications. Nat. Microbiol. 2, 17092 (2017).
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Competing interests
R.B. and P.H. are co-inventors on several patents related to CRISPR–Cas systems and
their various uses. R.B. is a co-founder and SAB member of Intellia Therapeutics and
Locus Biosciences, and a shareholder of Caribou Biosciences and DuPont. P.H. is an
employee of DuPont.

NATURE MICROBIOLOGY 2, 17092 (2017) | DOI: 10.1038/nmicrobiol.2017.92 | www.nature.com/naturemicrobiology 9

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