Crispr 3

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Mol Cell. Author manuscript; available in PMC 2015 April 24.
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Mol Cell. 2014 April 24; 54(2): 234–244. doi:10.1016/j.molcel.2014.03.011.
CRISPR-Cas systems: prokaryotes upgrade to adaptive

immunity
Rodolphe Barrangou1,# and Luciano A. Marraffini2
1Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University,
Raleigh, NC 27695, USA
2Laboratory of Bacteriology, The Rockefeller University, New York, NY 10065, USA
Summary
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), and associated proteins
(Cas) comprise the CRISPR-Cas system, which confers adaptive immunity against exogenic
elements in many bacteria and most archaea. CRISPR-mediated immunization occurs through the
uptake of DNA from invasive genetic elements such as plasmids and viruses, followed by its
integration into CRISPR loci. These loci are subsequently transcribed and processed into small
interfering RNAs that guide nucleases for specific cleavage of complementary sequences.
Conceptually, CRISPR-Cas shares functional features with the mammalian adaptive immune
system, while also exhibiting characteristics of Lamarckian evolution. Because immune markers
spliced from exogenous agents are integrated iteratively in CRISPR loci, they constitute a genetic
record of vaccination events and reflect environmental conditions and changes over time. Cas
endonucleases, which can be reprogrammed by small guide RNAs have shown unprecedented
potential and flexibility for genome editing, and can be repurposed for numerous DNA targeting
applications including transcriptional control.
Introduction
Bacteria dominate many natural habitats, including inhospitable environments and
challenging conditions that include predatory viruses. To survive, bacteria have developed
many ways to fend off invaders (Labrie et al., 2010), including the recently described
CRISPR system. CRISPR is an acronym for clustered regularly interspaced short
palindromic repeats. CRISPR loci contain short, partially palindromic DNA repeats that
occur at regular intervals and form loci that alternate repeated elements (CRISPR repeats)
and variable sequences (CRISPR spacers) (Figure 1). These peculiar loci were first observed
in 1987 (Ishino et al., 1987), but they received little attention until similar, idiosyncratic loci,
were described in microbial genome drafts (Jansen et al., 2002). These loci are typically
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Corresponding author: rbarran@ncsu.edu.
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Barrangou and Marraffini Page 2
flanked by accompanying CRISPR-associated (cas) genes. Their biological role remained

elusive until 2005, when three groups reported that the spacers were homologous to foreign
genetic elements, including viruses and plasmids (Bolotin et al., 2005; Mojica et al., 2005;
Pourcel et al., 2005). These reports lead to the hypothesis that CRISPRs might function as
an immune system (Makarova et al., 2006). Evidence for CRISPR-mediated immune
function quickly followed (Barrangou et al., 2007), and subsequent studies established that
CRISPR-mediated defense involves sequence-specific, RNA-mediated (Brouns et al., 2008)
targeting of mostly DNA (Marraffini and Sontheimer, 2008), and occasionally RNA (Hale et
al., 2009). Since then, many studies have delved in the molecular underpinnings of the
CRISPR-Cas system genetics, mechanisms, and applications.
Generally, CRISPR-Cas systems and their elements are hypervariable and differ broadly in
terms of occurrence, genes, sequences, number, and size across genomes. Indeed, CRISPR
repeats can vary widely (23–55 nt), though they are typically 28–37 nt, and their partially
palindromic nature allows them to form hairpin structures. Likewise, CRISPR spacers can
also vary in size (21–72 nt), though they are typically 32–38 nt. Quantitatively, arrays that
contain up to 588 repeats have been reported (in Haliangium ochraceum), but they are less
than 50 units in most cases. Likewise, although up to 19 distinct loci have been reported in
Methanocalcococcus, and 25 putative CRISPR loci have been suggested in Methanotorris

igneus, organisms typically contain 1–2 CRISPR loci (Grissa et al., 2007). According to the
database, CRISPRdb, CRISPRs occur in nearly half (1,126/2,480 ~45%) of bacterial
genomes and the large majority (125/150 ~83%) of archaea (Grissa et al., 2007).
CRISPR-Cas systems have been classified into three major Types, namely Type I, Type II
and Type III, and 12 subtypes, given their genetic content and structural and functional
differences (Makarova et al., 2011a; Makarova et al., 2013). The core defining feature of
CRISPR-Cas types and subtypes are the cas genes and the protein they encode, which are
highly genetically and functionally diverse, illustrating the many biochemical functions that
they carry throughout the different steps of CRISPR-mediated immunity. Noteworthy, the
RNA recognition motif (RRM) is widespread in many Cas proteins, and most of the Cas
families of proteins carry functional domains that interact with nucleic acids, such as DNA
binding, RNA binding, helicase, and nuclease motifs (Makarova et al., 2002; Makarova et
al., 2011a; Makarova et al., 2006; Makarova et al., 2011b; Makarova et al., 2013).
Genetically, cas1 and cas2 universally occur across types and subtypes, whereas cas3, cas9
and cas10 have been defined as the signature genes for Type I, Type II and Type III,
respectively. Phylogenetically, Type II systems have solely been identified in Bacteria, thus
far, and there is a bias for Type I systems in bacteria, and Type III systems in archaea and
hyperthermophiles.
Overall, CRISPR-Cas immune systems function in three steps. The first step is adaptation,
in which new spacers are acquired from exogenous nucleic acid into the CRISPR locus. The
adaptation step is followed by crRNA biogenesis, in which CRISPR arrays are transcribed
and processed into small interfering CRISPR RNAs (crRNAs). The final step is targeting, in
which crRNAs guide Cas nucleases for specific cleavage of homologous sequences (Figure
1). These steps have been described in a number of reviews published since 2008, including
several recent extensive or focused detailed reviews (Barrangou, 2013; Barrangou and

Horvath, 2012; Fineran and Charpentier, 2012; Marraffini, 2013; Reeks et al., 2013; Sorek
et al., 2013; Westra et al., 2012; Wiedenheft et al., 2012). Comparative analyses have
unraveled potential common ancestry between CRISPR-Cas system components and core
elements that define mobile genetic elements (notably transposases), as well as other defense
systems such as toxin-anti-toxin and restriction-modification systems (Makarova et al.,
2013).
Here, we provide an overview of CRISPR-based adaptive immunity in bacteria and archaea

and discuss the applications of CRISPR-Cas systems and their components.
CRISPR-Cas systems provide adaptive immunity

Although there are several innate immunity-like systems in bacteria, such as abortive
infection, receptor mutation, and restriction-modification, the recently characterized
CRISPR-Cas system has been described as an adaptive immune system, which provides
specific and acquired immunization against exogenic mobile genetic elements.
The first biological evidence that CRISPR-Cas systems have a role in adaptive immunity
was reported in 2007 when S. thermophilus CRISPR loci were shown to acquire novel
spacers derived from the invasive phage DNA (Barrangou et al., 2007). The acquisition of
phage DNA into the leader end of a CRISPR array led to sequence-specific, inheritable
immunity against phages bearing homologous sequences. Only a small proportion of the
population gained CRISPR encoded immunity, but this immunization, albeit infrequent,
provided a high level of resistance (Barrangou et al., 2007; Deveau et al., 2008; Levin et al.,
2013). Several studies of the S. thermophilus system have shown that most areas of the viral
genome are targeted, including both DNA strands, coding and non-coding sequences, and all
transcription modules. Nevertheless, a recent study showed that sampling of the viral
genome is biased (Paez-Espino et al., 2013), which might be due to DNA structural or
composition features. There is a danger that CRISPR systems can target host sequences.
Indeed, self-targeting is a very rare and lethal event(Paez-Espino et al., 2013). Given this
danger, CRISPRs must have a way to distinguish self from non-self, which is yet to be
characterized. Several subsequent studies in S. thermophilus confirmed this adaptive
immunity phenomenon, and characterized genetic elements involved (Deveau et al., 2008;
Horvath et al., 2008). A study in 2010 showed this CRISPR-Cas system can also vaccinate
cells against plasmid uptake by adaptive spacer acquisition (Garneau et al., 2010).
Noteworthy, spacers were acquired against antibiotic resistance genes, which could prevent
the uptake of any plasmid bearing complementary sequences.
Several studies of model CRISPR-Cas systems have also shown adaptive spacer uptake in E.
coli Type I-E systems (Datsenko et al., 2012; Swarts et al., 2012; Yosef et al., 2012) from
plasmid exposure, as well as in a Type III system from Sulfolobus solfataricus (Erdmann
and Garrett, 2012). Interestingly, in these studies, a phenomenon described as “priming”
showed that the first immunization events influence subsequent acquisition events, again
supporting the adaptive nature of these processes.
The sequence in the exogenous nucleic acid element corresponding to a CRISPR spacer has
been defined as a protospacer (Deveau et al., 2008), which is flanked by a system-specific

highly conserved CRISPR motif, subsequently renamed protospacer adjacent motif (PAM)
(Mojica et al., 2009) (Figure 2). PAMs are typically 2–5 nt highly conserved sequence
motifs immediately flanking one side of the protospacer (within 1–4 nt of one extremity).
These motifs have been identified in several Type I systems (where they primarily consist of
2–3nt motifs occurring on the 5′ end of the protospacer, Figure 2A) and many Type II
systems (where they range between 2–5nt and primarily occur on the 3′ end of the
protospacer, Figure 2B), while they are yet to be characterized in Type III systems (Mojica
et al., 2009; Sorek et al., 2013). PAMs have been implicated in both immunization
(sampling of the exogenous DNA for spacer uptake) (Paez-Espino et al., 2013) and targeting
(because PAM mutations preclude target cleavage) (Gasiunas et al., 2012; Jiang et al.,
2013a; Jinek et al., 2012; Sapranauskas et al., 2011; Sashital et al., 2012). This is consistent
with the preferential mutation of the PAM by viruses to escape CRISPR immunity
(Semenova et al., 2011; Sun et al., 2013).
Although we still do not fully understand how spacers are acquired and how new CRISPR
repeats are synthesized, Cas1 and Cas2 are involved. Spacer acquisition in Type I systems
require overexpression of cas1 and cas2 (Yosef et al., 2012), and acquisition is lost when
cas1 and cas2 are mutated (Datsenko et al., 2012). This is consistent with the sequence-
based co-evolutionary patterns that couple cas1 to CRISPR repeat sequences (Horvath et al.,
2009). Furthermore, cas1 and cas2 (and the proteins they encode) are dispensable for other
CRISPR-related processes such as crRNA biogenesis and targeting (Brouns et al., 2008;
Deltcheva et al., 2011; Hatoum-Aslan et al., 2014; Sapranauskas et al., 2011).
Biochemical studies of Cas1 and Cas2 have provided insights into their functional roles.
Cas1 binds tightly to dsDNA in a sequence-independent manner and both Cas1 and Cas2 are
metal-dependent endonucleases (Beloglazova et al., 2008; Nam et al., 2012; Samai et al.,
2010). Cas1 can cleave dsDNA into short fragments that might serve as precursors for novel
spacers (Han et al., 2009; Wiedenheft et al., 2009). Because Cas1 also has the ability to
resolve Holliday junctions and thus promote DNA integration and recombination events, it
is a noteworthy candidate for both novel spacer sampling and integration into the repeat-
spacer array.
Clearly, much of the mechanisms involved in spacer acquisition are unknown. The
molecular details involved in and responsible for recognizing non-self DNA, scanning for
candidate protospacers, generating a novel repeat, and polarized integration of the new
repeat-spacer unit into the CRISPR array are yet to be determined.
Lamarckian vs Darwinian evolution

CRISPR loci that are modified to provide immunity in one generation can be passed on to
the next generation. This heritability of acquired characteristics, together with the patterns of
CRISPR-Cas evolution, provide strong examples of Lamarckian evolution. (Koonin and
Makarova, 2013). Indeed, as aforementioned, there is a non-random bias in novel spacer
sampling and acquisition, which provides a phenotypic trait (phage resistance) that is
beneficial to the immunized host, and passed on to the surviving cell line. This contrasts
with the random and subsequently selected for nature of events assigned to Darwinian

phenomena. CRISPR loci are examples of bona fide Lamarckian evolution, especially given
the direct link between the acquired genotype (novel spacer integrated into the host genome)
and the corresponding phenotypic change (the ability to resist complementary sequences),
which is the essence of the Lamarckian paradigm (Koonin and Wolf, 2009). Furthermore,
the Lamarckian argument is supported by the fact that environmental cues (phage attack),
trigger heritable genetic changes (spacer integration) that provide adaptation (resistance to
the predatory phage). Lastly, the natural co-existence of a diversity of CRISPR genotypes
(Paez-Espino et al., 2013; Sun et al., 2013) supports the mathematically-predicted
Lamarckian nature of CRISPR genotype evolution in mixed populations (Haerter and
Sneppen, 2012). We should point out that some aspects of CRISPR-Cas systems are more
consistent with Darwinian evolution, notably the increased fitness of the variants that have
acquired novel spacers (Childs et al., 2012), and more precise insights into the type of
evolution that CRISPR systems provide to bacteria will be available as the molecular
mechanisms of spacer acquisition are elucidated.
RNA-guided Cas nucleases protect the host by destroying the invader’s

genome
The classification of CRISPR-Cas systems into three types primarily reflects the different
cas gene content of these systems. Since the Cas proteins are responsible for crRNA
biogenesis and the recognition and destruction of the invading nucleic acid, it is not
surprising that each CRISPR type displays a unique molecular mechanism of action (Figure
2).
Types I and III systems exhibit the most similarities (Figure 2A and C). Both systems rely
on the Cas6 family of endoribonucleases to cleave the repeat sequences of the crRNA
precursor to generate small crRNAs. Cas6 cleavage occurs typically 8 nucleotides upstream
of the 5′ end of the spacer sequence, generating crRNAs with 8 nucleotides of repeat
sequence at their 5′ end, known as the crRNA tag (Carte et al., 2008; Hatoum-Aslan et al.,
2011). In Type III CRISPR-Cas systems, however, crRNAs are further trimmed at the 3′
end by an unknown nuclease (Hale et al., 2008; Hatoum-Aslan et al., 2011) (Figure 2C).
Targeting in both Types is facilitated by a large, multi-subunit ribonucleoprotein complex.
The targeting complex in Type I systems is called Cascade (CRISPR-associated complex for
antiviral defense) (Brouns et al., 2008), which is formed by Cse1, Cse2, Cas7, Cas5, Cas6e
subunits in E. coli (Type IE) and Csy1, Csy2, Csy3 and Cas6f in Pseudomonas aeruginosa
(Type IF) (Wiedenheft et al., 2011). Nucleolytic cleavage of the target DNA is carried out
by a Cas3 family exonuclease, which is not a member of the complex (Sinkunas et al., 2013)
(Figure 2A). Two sequences in the target are essential for cleavage, the PAM and “seed”
sequences. In the E. coli Type IE system the PAM sequence consists of the three-nucleotide
motif AAG (Savitskaya et al., 2013; Swarts et al., 2012; Yosef et al., 2012), is located
immediately upstream of the protospacer and is recognized by the Cascade complex,
through direct contacts between Cse1 and the target DNA (Sashital et al., 2012). Because the
PAM sequences are absent from the repeats, this requirement provides a mechanism for
tolerance to ‘self’ sequences that are homologous, i.e. the spacers in the CRISPR array. The
“seed” sequence is a 6- to 8-nucleotide sequence within the guide crRNA which is strictly

complementary to the target sequence, (Semenova et al., 2011; Wiedenheft et al., 2011), and
enthalpically drives complementary base-pairing between the crRNA and the target, and
likely R-loop formation. It is hypothesized that the first event in target recognition are
transient, short-lived protein:DNA interactions between the Cascade complex and the PAM
sequence. This is followed by an attempt to hybridize the crRNA:DNA seed sequences,
most likely the rate-limiting step of the targeting reaction. Evidence for this model comes
from studies of Cas9 targeting (Sternberg et al., 2014) (see below). The PAM and seed
requirements for cleavage are readily exploited by mutant phages containing modifications
of either of these sequences, which prevent cleavage and CRISPR immunity (Barrangou et
al., 2007; Deveau et al., 2008).
The different Type III CRISPR-Cas systems seem to differ in the target nucleic acid: while
genetic data indicates that Type III-A CRISPR-Cas systems cleave DNA molecules in vivo
(Marraffini and Sontheimer, 2008), Type III-B CRISPR-Cas systems cleave RNA targets in
vitro (Hale et al., 2009) (Figure 2C). In both cases, targeting requires a large
ribonucleoprotein complex, the Cas10-Csm complex in Type III-A (Hatoum-Aslan et al.,
2013) and the CMR complex in Type III-B (Hale et al., 2009; Staals et al., 2013; Zhang et
al., 2012). Both targeting complexes possess the signature subunit Cas10, which most likely
harbors the nucleolytic activity of the complex (Hatoum-Aslan et al., 2014; Ramia et al.,
2013).
In contrast to Types I and III systems, Type II systems require minimal Cas machinery for
immunity (Figure 2B). For crRNA biogenesis, these systems utilize a trans-encoded
CRISPR RNA (tracrRNA), a small RNA that shares partial complementarity with CRISPR
repeats (Deltcheva et al., 2011). Pairing between the tracrRNA and the precursor crRNA
generates a double-stranded substrate that is cleaved by the host-encoded RNase III to
liberate the small crRNAs. These are further trimmed at their 3′ end by an unknown
nuclease. Cleavage of the DNA target in Type II systems is carried out by a single large
multidomain protein, Cas9. This is an RNA-guided double-stranded DNase with two
independent nuclease domains, HNH and RuvC, each of which cleaves one strand of the
target DNA (Gasiunas et al., 2012; Jinek et al., 2012). Recent studies have established that
formation of a complex with crRNA:tracrRNA drives Cas9 conformation changes that direct
target DNA binding and cleavage, in a PAM-dependent manner (Jinek et al., 2014;
Nishimasu et al., 2014; Sternberg et al., 2014).
Comparison between CRISPR-Cas and the mammalian adaptive immune

systems
To defend the host successfully, adaptive immune systems need to develop three essential
features: specificity, diversity, and memory. Specificity is required to recognize the
exogenous structures and organisms that need to be attacked, and distinguish them from
those that are to be tolerated. Diversity in the immune response is necessary to defend the
host from a large variety of pathogens and/or malignant elements, as to ensure survival to
exposure from a broad range of invasive agents. Finally, the generation of a memory of past
infections allows a more efficient reaction upon recurrent infections, and allows the host to
rapidly mount a response upon re-exposure. Both the adaptive branch of the mammalian

immune system (Murphy, 2012) and CRISPR-Cas immunity have these key features.
However, the molecular and cellular mechanisms that enable these features are different.
Below, we contrast these differences; for a more detailed comparison between both immune
systems we refer the readers to a recent review on this topic (Goren et al., 2012).
A fundamental distinction between both systems is the molecular nature of their target.
While mammalian immunity has to deal with a wide range of exogenous agents and types of
invasive cellular organisms that often display variable structural elements, the principal
infectious agents of prokaryotes are viruses and plasmids. In addition, whereas mammalian
immunity need to survey and protect distinct tissues within the host, unicellular prokaryotes
need to attack the cytoplasmic nucleic acids of the invader; i.e. within the context of a single
cell with a short lifespan. As a consequence of these differences, the molecules that provide
specificity to detect these targets are different. In the mammalian adaptive immune system,
antibodies (proteins) specifically recognize and bind three-dimensional antigens, namely
portions of proteins, sugar or nucleic acids present in the organisms and/or malignant cells
that need to be recognized. The proteinaceous nature of the antibodies allows them to
circulate throughout the body as free molecules or at the surface of lymphocytes. The
equivalent of the antibody/antigen pair in CRISPR immunity are the crRNA and the
protospacer sequence within the invading genome, which interact within the boundaries of
the prokaryotic cell. The mechanisms to achieve specificity are also fundamentally different.
While B cells undergo several rounds of maturation to select, in a Darwinian fashion, the
clones expressing the fittest antibodies for a particular antigen, CRISPR-Cas immune
systems directly acquire the protospacer sequences to produce a corresponding crRNA. This
relatively simple mechanism is possible due to the one-dimensional nucleic acid interaction
required to execute CRISPR immunity and sets the basis for the Lamarckian adaptation of
bacteria to their viruses provided by this immune system.
The specificity of the immune response is required to prevent the attack of ‘self’ elements
and ‘non-self’ commensal organisms. In mammals, the central tolerance mechanisms ensure
that newly developing B cells producing anti-self antibodies and T cells recognizing MHC-
presented self-peptides with high affinity are eliminated in the bone marrow and in the
thymus, respectively. Peripheral tolerance mechanisms, on the other hand, inactivate mature
lymphocytes to promote tolerance to ‘non-self’ elements, such as the gut commensal
microbiota and innocuous dietary proteins. In the CRISPR immune response, tolerance to
‘self’ is required to prevent the targeting of the spacer sequences within the CRISPR array,
which are, by nature, complementary to the crRNAs they encode. This is achieved through
recognition by the Cas machinery of features in the spacer flanking sequences that are not
present in the flanking sequences of bona-fide protospacers (see above). Another mechanism
for tolerance to ‘self’ is required to prevent the acquisition of spacer sequences from the
bacterial chromosome. Whether and how CRISPR-Cas systems distinguish self DNA from,
for example, phage DNA during the acquisition phase is yet to be determined. Tolerance to
‘non-self’ has not been observed for CRISPR immunity so far. Beneficial plasmids and
prophages (bacteriophages that integrate into the host chromosome) could be considered
commensal elements of bacteria and an immune response towards these elements would be
detrimental for the host. Experimental evidence, however, indicates that an active CRISPR-

Cas system and its target cannot co-exist in the same cell (Jiang et al. 2013b) and therefore
tolerance of plasmids (Jiang et al., 2013b) and prophages (Edgar and Qimron, 2010; Nozawa
et al., 2011) is not possible. Perhaps the regulation of CRISPR immunity at the
transcriptional level (Pul et al., 2010; Westra et al., 2010; Yosef et al., 2011) could provide
an ‘on-off’ switch for the tolerance of prokaryotic foreign genetic elements.
In mammalian immune systems, diversity is achieved through the generation of many

different antibodies through V(D)J recombination and somatic hypermutation of light chain
genes. This ensures that virtually any target can be recognized. After the infection is clear, a
subpopulation of the lymphocytes that produce the most effective antibodies become
memory cells, ready to proliferate and attack the pathogen during subsequent exposures. As
mentioned previously, CRISPR-Cas systems acquire spacers directly from the invading
genome, at the same time capturing and storing the diversity and memory of the CRISPR
immune response into the CRISPR array. It is suspected that any viral and plasmid DNA
sequence can be acquired, as long as they are adjacent to a proper PAM sequence, and in
principle the CRISPR immune response can deal with the great diversity of prokaryotic
viruses and plasmids. It is yet to be established, however, whether CRISPR immunity can
also defend the host from RNA phages (in this case the acquisition of spacers will require
reverse transcription), which represent a substantial proportion of the prokaryotic viruses

(Zinder, 1980). Importantly, in contrast to the antibody diversity and memory generated by
the mammalian immune system during the life span of the individual, most of the spacers
acquired during CRISPR immunity are inherited by the progeny after bacterial duplication
(though some can be lost, presumably during DNA replication of the repeat sequences
through polymerase slippage). The inheritable nature of CRISPR-Cas systems is a major
advantage for the host’s progeny, and a major difference between these two immune
systems. A final contrast regards how memory is generated in both systems. In mammals,
memory lymphocytes can rise after either infection or vaccination. For CRISPR, it remains
to be determined whether spacers can be acquired and expressed during infection, before the
infecting phage kills the cell, or from occasional defective viruses that inject their DNA into
the prokaryotic host (a process analogous to vaccination).
Applications of CRISPR-Cas systems

The genetic polymorphism of cas genes, together with the functional diversity of the
proteins they encode, and in combination with the features and natural functions of
CRISPR-Cas systems, have set the stage for a broad array of applications, both exploiting
their native roles, and by re-purposing their functional features.
Using CRISPR diversity for genotyping

Originally, the first application of CRISPR loci was exploiting CRISPR repeat occurrence
and number diversity to type Mycobacterium and Yersinia (Pourcel et al., 2005) isolates, as
defined by spacer oligo typing (spoligotyping) (Groenen et al., 1993). Spacer content was
subsequently used for genotyping bacteria of industrial importance, and providing insights
into the common origin of strains as defined by the conservation of ancestral spacers
(Barrangou, 2012). Indeed, polarized spacer addition at the leader end of the CRISPR locus
provides a genetic tape record of immunization events, and a basis for genetic tracking of

the genetic trajectory of a strain. CRISPR loci have been used for genotyping of several
bacteria including Mycobacterium, Yersinia, Corynebacterium, Pseudomonas, Legionella,
Streptococci, Escherichia, Salmonella, and Lactobacillus, among others (Barrangou and
Horvath, 2012; Shariat et al., 2013). This approach can also be used to determine the
relatedness of strains that are of pathogenic interest, notably in food outbreak investigations
such as E. coli and Salmonella (Shariat et al., 2013).
Beyond single strain phylogeny, CRISPR loci provide a basis for probing complex
ecological populations and determine the diversity of both host and virus populations in
complex systems, as documented in natural habitats (Andersson and Banfield, 2008;
Heidelberg et al., 2009; Held and Whitaker, 2009; Tyson and Banfield, 2008) and human
samples (Pride et al., 2011). Extensive analyses of CRISPR sequences using metagenomic
deep sequencing technologies can also provide critical insights into co-evolutionary
dynamics, and the evolutionary trajectories of both host and virus populations, as the arms
race unfolds (Levin et al., 2013; Paez-Espino et al., 2013; Sun et al., 2013). Indeed, CRISPR
has been shown to be a major force shaping viral genome evolution in some systems, in
which SNPs, INDELs and recombinations specifically allow viruses to circumvent CRISPR-
encoded adaptive immunity in the host. Nevertheless, the potential of CRISPR for broad
genotyping or epidemiological surveys, clinical diagnostics, and industrial surveillance will

necessitate validation across a diverse set of genera and species of interest.
Building resistance to viruses in industrial cultures

From an industrial standpoint, there is tremendous need of and potential for building phage
resistance in industrially relevant hosts that historically have been, or could be at risk of
phage attacks. Keeping in mind the absurd scale of some industrial bioprocessing plants that
rely on bacteria to generate bioproducts of interest (therapeutics, enzymes) and foods (dairy
products), the risk of phage attack and impact of phage outbreaks can equate to financially
catastrophic and significant losses. The original discovery of CRISPR adaptive immunity in
the dairy starter culture Streptococcus thermophilus has already been harnessed to obtain
phage-resistant mutants to formulate dairy starter cultures with improved sustainability and
lifespan for perennial exploitation in the food industry (Barrangou and Horvath, 2012).
Noteworthy, it has been shown that iterative build up of viral resistance is efficient, using a
set of genetically diverse phages to optimize the breadth and depth of phage resistance
(Barrangou et al., 2013; Barrangou and Horvath, 2012). Practically, the unique set of
acquired spacers can also serve as a genetic tag for valuable proprietary strains.
Notwithstanding early adoption of CRISPR-based lytic phage resistance in dairy cultures,
native systems may be broadly used in other industrial bacteria. Also, the ability to readily
package and heterologously transfer CRISPR-Cas systems in prokaryotes (Sapranauskas et
al., 2011) and even eukaryotes (Cong et al., 2013; Mali et al., 2013b) illustrate the
widespread potential of CRISPR-mediated viral resistance, either naturally, or using genetic
engineering given the ability to design and synthetize spacers to re-program Cas nucleases
(Sapranauskas et al., 2011). The viral resistance upside is illustrated by a recent report
showing excision of HIV-1 provirus in human (Ebina et al., 2013), further highlighting the
broad promise of this technology for viral immunity beyond the natural hosts of CRISPR-
Cas systems.

Beyond viral resistance, CRISPR-mediated adaptive immunity has potential to vaccinate

bacterial strains against the uptake and dissemination of undesirable genetic elements such
as plasmids that carry antibiotic resistance genes (Garneau et al., 2010) and virulence traits
(Bikard et al., 2012). Likewise, because CRISPR can be used to target mobile genetic
elements, there is potential to use CRISPR to target transposons and other mobile elements
that contribute to genome plasticity as to ensure the maintenance, integrity and stability of
the genomes of industrial workhorses widely used in the biotechnology, pharmaceutical and
bioproducts industries.
Genome Engineering
The early models for CRISPR-Cas systems postulated that interference was RNA-mediated
and protein-dependent, akin to the RNA interference mechanism of eukaryotes (Makarova et
al., 2006). It was later established that DNA is the primary target of CRISPR-Cas systems
(Marraffini and Sontheimer, 2008), and that interference occurs through sequence-specific
target DNA cleavage (Garneau et al., 2010; Marraffini and Sontheimer, 2008). These
findings arguably laid the foundation for the subsequent use of these systems as
programmable nucleases, with many inherent biotechnological applications. The detailed
characterization of CRISPR immunity that followed, particularly of the Type II, Cas9-
mediated immunity (Deltcheva et al., 2011; Gasiunas et al., 2012; Jinek et al., 2012;
Sapranauskas et al., 2011), led to the realization of the potential for this enzyme in genome
engineering. 2013 has seen an explosion of reports that use the RNA-guided programmable
feature of Cas9 for the development of powerful technologies for genome editing, genetic
screens and modulation of gene expression (Pennisi, 2013), which we describe briefly
below.
Engineering genomes with Cas9—While Type I and III CRISPR-Cas systems provide
RNA-guided nuclease activity, this is achieved in the context of a large, multimeric, crRNA-
Cas ribonucleoprotein complex (Figure 2) that complicates the development of a molecular
tool. In contrast, Type II systems only rely on a single endonuclease which generates
predictable dsDNA breaks into the target sequence, namely Cas9 (Sapranauskas et al.,
2011). In truth, Cas9 requires the participation of an “enabling RNA”, tracrRNA, as well as
RNaseIII, to load the crRNA guide (Deltcheva et al., 2011) (see above and Figure 2B) and
the crRNA and tracrRNA to cleave its target (Gasiunas et al., 2012; Jinek et al., 2012). Both
of these requirements can be bypassed by the use of a chimeric RNA guide, also known as
single-guide RNA (sgRNA): a fusion between the tracrRNA and crRNA that maintains the
guide properties of the crRNA as well as the tracrRNA:crRNA secondary structures that are
critical for Cas9 cleavage (Jinek et al., 2012). This technological improvement was
fundamental towards establishing Cas9 as an ideal RNA-guided dsDNA nuclease, and has
led many researchers to exploit it as a genetic engineering tool, in the same way ZFNs and
TALENs have been used in the past (Wood et al., 2011).
In a bacterial host, Cas9 cleavage of genomic sequences leads to cell death, presumably due
to the introduction of chromosomal lesions that can only be repaired with high fidelity, thus
re-introducing the target (Bikard et al., 2012; Jiang et al., 2013a). The absence of target
mutations that could escape Cas9 cleavage are thought to be the consequence of the lack of

non-homologous end joining (NHEJ) repair mechanisms in most bacteria (Shuman and
Glickman, 2007). This phenomenon has been exploited to use Cas9 as a powerful and
efficient counter-selection tool to introduce specific mutations in bacteria, essentially using
CRISPR-based self targeting for directed mutagenesis (Bikard et al., 2012). In contrast,
dsDNA breaks in mammalian DNA are partially repaired by the INDEL-generating NHEJ
pathway (Figure 3) (Deriano and Roth, 2013). Therefore expression of S. pyogenes Cas9 and
an appropriate sgRNA can be used to introduce INDELs and knock-out virtually any gene,
provided that it contains a the corresponding PAM sequence (GG in this case, or CC on the
complementary strand) (Cong et al., 2013; Jinek et al., 2013). The high efficiency of Cas9-
mediated INDEL generation has been used to perform forward genetic screens in which
cells expressing the nuclease are transduced with a lentivirus expressing a library of sgRNAs
that target each gene of the cell. Analysis of the sgRNA sequences present in cells screened
or selected for a particular phenotype is used to identify the candidate genes responsible for
this phenotype (they are presumably mutated) (Shalem et al., 2013; Wang et al., 2013b). In
addition, dsDNA breaks stimulate homologous recombination in mammalian cells (Smih et
al., 1995), thus the introduction of a suitable template that can recombine with the target
sequence can be employed to generate site-directed, single-nucleotide substitutions (Figure
3) (Cong et al., 2013; Mali et al., 2013b). Moreover, the co-expression of Cas9 and several
RNA guides can be exploited for the generation of multiple mutations in one single
multiplexing step (Cong et al., 2013; Wang et al., 2013a).
An unresolved issue for this technology is the frequency of off-target effects, i.e. the
probability of Cas9 cleavage of a sequence partially homologous to the sgRNA. While this
undesirable effects are known to occur (Fu et al., 2013), researchers have developed
strategies to overcome them with the use of a RuvC active site mutation that converts Cas9
into a nickase, optimally exploited using two paired guides that direct nicking at two
adjacent sites only in the intended target sequence (Mali et al., 2013a; Ran et al., 2013).
Despite this caveat, the use of Cas9-directed genome editing has been applied very
successfully in a broad range of hosts and cell lines, including in human cells, mice, rats,
zebrafish, plants, flies, nematodes, yeast, bacteria and many other organisms (Pennisi,
2013), creating a revolution in molecular biology arguably comparable to the introduction of
restriction endonucleases.
Control of gene expression using dCas9—With the mutation of both the RuvC and
HNH nucleolytic active sites (Gasiunas et al., 2012; Jinek et al., 2012), Cas9 can be
converted into a RNA-guided dsDNA binding protein, known as dCas9. This feature has
been exploited to repress or activate gene expression in bacteria and mammalian cells. In
both organisms, repression can be achieved by directing dCas9 to bind promoter sequences,
thus interfering with RNA polymerase (RNAP) transcription initiation, or bind sequences
within the open reading frame (particularly the template strand) and block transcription
elongation (Figure 3) (Bikard et al., 2013; Qi et al., 2013). Activation of gene expression, on
the other hand, requires the fusion of Cas9 to an activation domain. In bacteria, Cas9 can be
fused to the omega (ω) subunit of RNAP (rpoZ) in an ΔrpoZ background in Escherichia
coli. The dCas9-ω fusion can then be directed to promoter sequences using an appropriate
guideRNA to recruit RNAP and activate transcription (Bikard et al., 2013). In mammalian

cells, dCas9 can be fused to transcriptional activators such as VP64 and the p65 activator
domain (Figure 3) (Gilbert et al., 2013a; Maeder et al., 2013; Mali et al., 2013a). The
addition of other functional domains to dCas9 can expand the uses of the programmable
DNA binding capability of this enzyme. For example, the KRAB and Kox1 chromatin-
modifying domains can be used to attain stronger repression (Gilbert et al., 2013a), and GFP
can be used to visualize specific loci within the chromosome (Chen et al., 2013; Malina et
al., 2013). The fusion of other domains, such as recombinases, methylases and histone-
modifying enzymes will undoubtedly create new opportunities for this technology (Figure
3).
A perspective on the future of CRISPR immunity

There are many useful applications for CRISPR-Cas immunity and its biological
significance is no less exciting. For example, phages can acquire CRISPR-Cas systems (in
their genome) to specifically target host defense systems, an evolutionary puzzling turn of
events (Seed et al., 2013). There are also phages carrying genes that inhibit CRISPR
immunity (Bondy-Denomy et al., 2013). In the pathogen Francisella novicida, Cas9 can
repress an endogenous lipoprotein gene to promote pathogenesis by preventing the host
proinflammatory response against this lipoprotein (Sampson et al., 2013). CRISPR-Cas

systems have also been shown to acts as barriers against horizontal gene transfer (Bikard et
al., 2012; Jiang et al., 2013b; Zhang et al., 2013), a fact that highlights the importance of
CRISPR immunity in the evolution of prokaryotes. One may accordingly wonder: what is
next for the CRISPR field?
Undoubtedly, the translation of Cas9-based genome editing technologies into medical

applications, most notably gene therapy, has the potential to make a significant impact on
human health (provided that the off-target effects are reduced to acceptable levels). Recent
advances in understanding the biochemical nature of Cas9 targeting (Sternberg et al., 2014),
together with structural insights into Cas9:sgRNA complex formation (Jinek et al., 2014;
Nishimasu et al., 2014) will lead to the development of improved molecular biology tools. In
addition, a cursory look at the CRISPR intellectual property landscape, publication pace, and
citation rate reflect a widespread awareness of the potential of CRISPR-Cas systems and
suggests that they will rapidly be routinely used in many, if not most, molecular biology
laboratories. Therefore the accelerating pace of industrial exploitation, commercialization
and financial investment(s) is setting the stage for a sustainable CRISPR revolution.
In addition to the potential of CRISPR-Cas9 for genome surgery in eukaryotic cell lines, the
native role of CRISPR-Cas systems in providing adaptive immunity has a dramatic impact
on microbial communities and their predators in most habitats (Andersson and Banfield,
2008; Heidelberg et al., 2009; Held and Whitaker, 2009; Tyson and Banfield, 2008), and
their true impact is yet to be defined on a global basis. If adaptive immunity in characterized
model systems is representative of the overall impact of CRISPR-Cas systems on the host-
virus population co-evolutionary dynamics and genome trajectories, their role in the
composition, evolution and ecology of microbial communities in nature is and will continue
to be exciting. We also anticipate that additional applications and novel insights will stem

from further characterization of CRISPR model systems, their genetic elements, functional
modules and mechanism of action.
Acknowledgments
RB is supported by start up funds from North Carolina State University. LAM is supported by the Searle Scholars
Program, the Rita Allen Scholars Program, an Irma T. Hirschl Award, a Sinsheimer Foundation Award and a NIH
Director’s New Innovator Award (1DP2AI104556-01). We are grateful to Daniel Mucida (The Rockefeller
University) for critical reading of the manuscript. The authors would like to thank their many colleagues and
collaborators in the CRISPR field for their insights into these fantastic molecular systems.
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Highlights
• CRISPR-Cas systems provide DNA-encoded and RNA-mediated adaptive
immunity in bacteria
• Immunization occurs through uptake of DNA from invasive genetic elements

for sequence-specific vaccination
• Sequence-specific immunity is mediated by small interfering RNAs that guide

endonucleases for interference via DNA cleavage
• RNA-guided CRISPR nucleases have important applications, most notably for

the engineering of eukaryotic genomes.

Figure 1. The three stages of CRISPR immunity

CRISPR loci contain clusters of repeats (black diamonds) and spacers (colored boxes) that
are flanked by a “leader” sequence (L) and CRISPR-associated (cas) genes. During
adaptation new spacers derived from the genome of the invading virus are incorporated into
the CRISPR array (Barrangou et al., 2007) by an unknown mechanism. The synthesis of a
new Repeat is also required. During crRNA biogenesis a CRISPR precursor transcript is
processed by Cas endoribonucleases within repeat sequences to generate small crRNAs
(Brouns et al., 2008). During targeting the match between the crRNA spacer and target
sequences (complementary protospacer) specifies the nucleolytic cleavage (red cross) of the
invading nucleic acid (Garneau et al., 2010; Gasiunas et al., 2012; Jinek et al., 2012).

Figure 2. Mechanism of crRNA biogenesis and targeting in the three Types of CRISPR-Cas
systems
Black arrowheads, primary processing sites of the crRNA precursor (pre-crRNA) to liberate
intermediate crRNAs (int-crRNA). White arrowhead, further processing of the int-crRNA to
yield mature crRNAs (mat-crRNA). Green line, target sequence (same sequence as crRNA
spacer). Purple line, PAM (Mojica et al., 2009). (A) In Type I systems, primary processing
of the pre-crRNA is achieved by the Cas6 endoribonuclease within the Cascade complex
(Brouns et al., 2008). Cleavage occurs at the base of the stem-loop formed by the repeat
RNA to release mat-crRNAs. The Cascade recruits the Cas3 nuclease to nick the DNA
strand complementary to the proto-spacer, immediately downstream of the region of
interaction with the crRNA spacer (Sinkunas et al., 2013). (B) In Type II systems primary
processing requires the annealing of the tracrRNA to the repeat sequences of the pre-crRNA
and the subsequent cleavage of the dsRNA by the host RNase III (Deltcheva et al., 2011).
Primary processing occurs in the contex of Cas9 and it is followed by the trimming of the
5′-end repeat and spacer sequences of the int-crRNA to yield mat-crRNAs. Target cleavage
requires the crRNA, the tracrRNA and the RuvC and HNH domains of Cas9, each of which
cleaves one DNA strand of the proto-spacer region, 3-nt upstream of the PAM (Gasiunas et
al., 2012; Jinek et al., 2012). (C) In Type III systems Cas6 cleaves the pre-crRNA to
generate int-crRNAs that are incorporated into a Cmr/Cas10 or Csm/Cas10 complex, where
further maturation occurs through the trimming of 3′-end sequences (Hale et al., 2012; Hale
et al., 2009). While genetic evidence indicates that III-A subTypes cleave target DNA
sequences (Hatoum-Aslan et al., 2014), biochemical data suggests that subType III-B cleave
RNA molecules (Hale et al., 2009).

Figure 3. Cas9-based genetic applications

Wild-Type Cas9 loaded with a single guide RNA (sgRNA) generates dsDNA breaks that
can be used to introduce target mutations. Chromosomal breaks can be repaired by non-
homologous end joining (NHEJ), creating indels (Δ) that introduce knock-out frameshift
mutations. If a sequence homologous to the Cas9 target is provided (the editing template;
either linear dsDNA or a short oligonucleotide), the break can be repaired by homologous
recombination (Cong et al., 2013; Mali et al., 2013b). In this case, site-specific mutations in
the editing template are can also be incorporated in the genome. A catalytically dead Cas9
(dCas9, containing mutations in both the RuvC and HNH actives sites) can be used as an
RNA-guided DNA binding protein that can repress both transcription initiation when bound
to promoter sequences or transcription elongation when bound to the template strand within
an open reading frame (Bikard et al., 2013; Qi et al., 2013). dCas9 can also be fused to
different functional domains to bring enzymatic activities and/or reporters to specific sites of
the genome (Gilbert et al., 2013b).

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Mol Cell. 2014 April 24; 54(2): 234–244. doi:10.1016/j.molcel.2014.03.011.

CRISPR-Cas systems: prokaryotes upgrade to adaptive

© 2014 Elsevier Inc. All rights reserved.

flanked by accompanying CRISPR-associated (cas) genes. Their biological role remained

Methanocalcococcus, and 25 putative CRISPR loci have been suggested in Methanotorris

Mol Cell. Author manuscript; available in PMC 2015 April 24.

Here, we provide an overview of CRISPR-based adaptive immunity in bacteria and archaea

CRISPR-Cas systems provide adaptive immunity

Mol Cell. Author manuscript; available in PMC 2015 April 24.

Lamarckian vs Darwinian evolution

Mol Cell. Author manuscript; available in PMC 2015 April 24.

RNA-guided Cas nucleases protect the host by destroying the invader’s

Mol Cell. Author manuscript; available in PMC 2015 April 24.

Comparison between CRISPR-Cas and the mammalian adaptive immune

Mol Cell. Author manuscript; available in PMC 2015 April 24.

Mol Cell. Author manuscript; available in PMC 2015 April 24.

In mammalian immune systems, diversity is achieved through the generation of many

reverse transcription), which represent a substantial proportion of the prokaryotic viruses

Applications of CRISPR-Cas systems

Using CRISPR diversity for genotyping

Mol Cell. Author manuscript; available in PMC 2015 April 24.

genotyping or epidemiological surveys, clinical diagnostics, and industrial surveillance will

Building resistance to viruses in industrial cultures

Mol Cell. Author manuscript; available in PMC 2015 April 24.

Beyond viral resistance, CRISPR-mediated adaptive immunity has potential to vaccinate

Mol Cell. Author manuscript; available in PMC 2015 April 24.

Mol Cell. Author manuscript; available in PMC 2015 April 24.

A perspective on the future of CRISPR immunity

proinflammatory response against this lipoprotein (Sampson et al., 2013). CRISPR-Cas

Undoubtedly, the translation of Cas9-based genome editing technologies into medical

Mol Cell. Author manuscript; available in PMC 2015 April 24.

Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P.

Mol Cell. Author manuscript; available in PMC 2015 April 24.

Mol Cell. Author manuscript; available in PMC 2015 April 24.

Horvath P, Romero DA, Coute-Monvoisin AC, Richards M, Deveau H, Moineau S, Boyaval P,

Mol Cell. Author manuscript; available in PMC 2015 April 24.

Bacterial Pathogens. PLoS Pathog. 2013; 9:e1003765. [PubMed: 24348245]

Mol Cell. Author manuscript; available in PMC 2015 April 24.

Mol Cell. Author manuscript; available in PMC 2015 April 24.

Mol Cell. Author manuscript; available in PMC 2015 April 24.

• Immunization occurs through uptake of DNA from invasive genetic elements

• Sequence-specific immunity is mediated by small interfering RNAs that guide

• RNA-guided CRISPR nucleases have important applications, most notably for

Mol Cell. Author manuscript; available in PMC 2015 April 24.

Figure 1. The three stages of CRISPR immunity

Mol Cell. Author manuscript; available in PMC 2015 April 24.

Mol Cell. Author manuscript; available in PMC 2015 April 24.

Figure 3. Cas9-based genetic applications

Mol Cell. Author manuscript; available in PMC 2015 April 24.

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