MB3016 Literature Review

Title: CRISPRs, the new antiviral defense

system in prokaryotes.

Student’s name: Marcas O Muineachain

Student number: 106003290

Supervisor’s name: Dr. Douwe van Sinderen

INTRODUCTION

Prokaryotes and viruses are ubiquitous on earth, with viruses

believed to represent the most abundant biological entity on
our planet (Breitbart & Rohuer., 2005). Quantitatively, the
dominant predator – prey interaction in the biosphere is the
battle between bacteria and their viruses (Bacteriophages,
hereafter referred to as phages) (Young, R.F., Science vol
321., 2008). According to one estimate, 20% of the bacteria
on earth are lysed by phages every day and therefore phages
have a significant effect on microbial ecology and the
evolution of bacterial genomes (Chibani – Chennoufi et al.,
2004) To effectively fight this battle with phages, bacteria
proliferate and avoid extinction by multiple innate phage –
resistance mechanisms which target various steps of the phage
life cycle, such as blocking DNA injection, use of restriction
enzymes to degrade the incoming DNA, and abortive
infection, where phage replication is inhibited by the
premature death of the cell, thereby limiting the spread of
infectious phage particles to other bacteria (Sturino &
Klaenhammer., 2006).

The subject of this review is the recently discovered

Clustered, Regularly Interspaced Short Palindromic Repeat
(CRISPR) system, which was first shown by Barrangou and
colleagues to confer acquired resistance against viruses in
prokaryotes (bacteria and archaea) (Barrangou et al., 2007).
CRISPRs are widely distributed, as they are present in the
genomes of approximately 40% of bacteria and almost all
archaea (Jansen et al., 2002). A key feature of the CRISPR
system, which acts at the RNA level, is the arrays (loci) of
short direct repeats, separated by non – repetitive spacer
sequences which are approximately the same length.
Important additional features of the system include CRISPR –
associated (CAS) genes and a leader sequence (Godde &
Bickerton., 2006).
REPEATS, SPACERS, THE LEADER, AND CAS
GENES– THE STRUCTURAL COMPONENTS
OF THE CRISPR SYSTEM.

Figure 1. Three integral components of a CRISPR locus. Direct repeat sequences (repeats),
separated by intervening spacer sequences (spacers) with the leader sequence attached to the
5’ end of the first repeat. See text below for a detailed explanation. (figure from the
CRISPRfinder program online at http://crispr.u-psud.fr.)

The repeated sequences (repeats) are 24 – 47 bp and are

usually specific to a given CRISPR locus. Repeats are almost
always identical in a single locus with regard to the size and
sequence and it is the sequence of the repeat which normally
defines CRISPRs (Jansen et al., 2002). The number of repeats
per locus depends on the organism in question, with a
minimum of 2 and the current maximum is 249 repeats in
Verminephrobacter esiniae (Grissa et al., 2007) The terminal
repeat is normally degenerate (GAAA) at its 3’ end (Horvath
et al., 2008). Some repeats may differ slightly from the others
because of single nucleotide polymorphisms, but the typical
repeat sequence is understood to be the most frequent
sequence within a certain CRISPR locus (Horvath et al.,
2008).

Although the repeats differ between species, they are highly

conserved, and can be clustered based on the similarity
between the sequences into at least 12 significant groups
(Kunin et al., 2007). A recent hypothesis in the literature is
that the palindromes of some of the larger groups are what
contributes to the RNA secondary structure of the repeat
(post-transcription) (hence the word ‘Palindromic’ in the
acronym CRISPRs) (Kunin et al., 2007).
It has also been suggested by the same researchers that the
conserved 3’ terminus (GAAA) acts as a binding site for one
or more of the CRISPR associated (Cas) proteins (Kunin et
al., 2007).

Spacers are the sequences flanked by two consecutive

CRISPR repeats and are of constant and similar length,
typically 26 – 72 bp depending on the species or CRISPR
locus in question (Horvath et al., 2007). Figure 2 shows a
repeat (29 bp) and a spacer (32 bp) from one of the CRISPR
loci from E.coli K12.

Figure 2. A repeat and a spacer sequence from an E.coli K12 CRISPR locus. Convergently
pointing arrows mark a palindrome. (Adapted from Bolotin et al., 2008).

The spacers are almost always unique except in rare instances

where segmental duplications may occur (Grissa et al., 2007).
Unlike the direct repeats, which are highly conserved, the
number and sequence of the spacers show great diversity,
even among strains of the same species (Deveau et al., 2007).
Sequence searches of the multiple CRISPR spacers in a large
variety of bacteria revealed that many spacers show homology
(100% sequence identity) to sequences in genes commonly
found in phages and other extrachromosomal elements such as
plasmids (Mojica et al., 2005., Bolotin et al., 2005) and it has
also been demonstrated that bacteria acquire new spacers in
response to phage predation (Barrangou et al., 2007., Horvath
et al, 2008., Deveau et al., 2008). These experimental
observations have lead to the hypothesis that these spacers are
derived from the phage genome and confer immunity to the
bacteria (Barrangou et al., 2007).

The CRISPR leader consists of a sequence of 200 - 350 bp

found adjoining the 5’ end of the first repeat. The leader is A-
T rich and non-coding (Jansen et al., 2002). Like repeats,
leaders are not usually conserved among species because they
lack an open reading frame, however research has shown that
when several CRISPR loci are located in the same
chromosome, then their leaders can be conserved (Bult et al.,
1996., Klenk et al., 1997., Smith et al., 1997). Because the
leader is located directly upstream of the first repeat, it is
believed to act as a promoter for CRISPR repeat-spacer unit
transcription (Jansen et al., 2002., Lillestol et al., 2006).

CRISPR associated (Cas) genes are always found adjacent

to CRISPR loci (Figure 3) and together they constitute a
microbial immune system (Mojica et al., 2005, Barrangou et
al., 2007., Brons et al., 2008).

Figure 3. Diagram of a typical CRISPR locus, showing attached CAS genes.

These genes encode a wide variety of proteins and a recent
comprehensive bioinformatic analysis of the Cas system has
enabled the proteins to be classified into 45 families (Haft et
al., 2005., Makarova et al., 2006). The CRISPR systems have
been categorized into 8 subtypes, with each subtype
containing 2 to 6 different subtype-specific Cas genes along
with six core cas genes (Cas 1- 6) that are associated with
numerous subtypes, in particular Cas1 which is the universal
marker of the CRISPR system found in all CRISPR systems
except for Pyrococcus abyssii (Makarova et al., 2006). The
functions of the proteins which these genes encode is
described in table 1 below.
Cas1 Cas2 Cas3 Cas4 Cas5 Cas6
Nuclease Endoribonucleas Helicase Exonucleas Unknown Possible
(An enzyme e (An enzyme e (Function has RAMP
that degrades which not yet been
(A hydrolase enzyme) (An enzyme that (Believed to
nucleic acid separates two determined)
cleaves be a member
molecules) annealed
nucleotides one of the repair
nucleic acid
at a time from associated
strands)
the end of a mysterious
polynucleotide protein
chain) family)

Table 1. Cas genes and the functions of the proteins they encode (Self –constructed table,
Credits for the information: (Haft et al., 2006, Makarova et al., 2006, Beloglazova et al.,
2008))
THE MECHANISM OF ACTION: How CRISPRs
And Cas Proteins Confer Defense Against
Infection By Providing Acquired Resistance
Against Phages.
Recent studies have confirmed the hypothesis that CRISPRs
in association with the physically attached genes of Cas
proteins confer defense against infection by providing
acquired resistance against phages and the understanding of
the mechanism of this prokaryotic immune system has also
progressed substantially.

Barrangou and colleagues were the first to experimentally

demonstrate that in response to phage challenge, bacteria
integrate new spacers which are derived from phage genomic
sequences, and this confers resistance against infection
(Barrangou et al., 2007).
After infecting Streptococcus thermophilus with two distinct
but closely related phages (Phage 858 and 2972, isolated from
industrial yoghurt samples), they recovered nine phage-
resistant mutants. The CRISPR loci of these mutants was then
analysed and sequencing of the CRISPR1 locus showed that
each of the phage-resistant mutants had independently
obtained between one and four additional repeat-spacer units,
all polarized towards the leader end of the locus. Sequence
analysis of these new spacers showed that they were derived
from the genome of the infecting phage. The mutant was
found to be phage resistant if a spacer was homologous to the
phage sequence. However when nucleotide changes
(polymorphisms) were observed between the spacer and the
phage sequence, the spacer did not confer resistance and the
bacteria were observed to be phage-sensitive. The spacers
which provided resistance were then inserted into the CRISPR
locus of a phage sensitive S.thermophilus, and this strain was
then observed to become immune to phage infection. When
they then deleted the acquired spacers, the strain was again
sensitive to phage infection (Barrangou et al., 2007).
Altogether, these results showed that the inclusion of a
CRISPR spacer identical to a phage sequence confer
resistance against phages that contain this particular sequence.
Barrangou and colleagues also demonstrated the importance
of Cas genes by showing that when a certain Cas gene (Called
Cas5 by Barrangou and colleagues) was inactivated in a
phage- resistant strain of S.thermophilus, immunity is lost and
the mutant is no longer phage resistant (Barrangou et al.,
2007).
Brouns and colleagues (Brouns et al., 2008) recently
demonstrated using E.coli K12 that virus-acquired sequences
contained in CRISPRs are used by Cas proteins from the host
to mediate an antiphage response which effectively combats
infection. Following transcription of the CRISPR, a complex
of Cas proteins, which the authors termed “Cascade” (Brouns
et al., 2008), cleaves a CRISPR RNA precursor in each repeat
and keeps the cleavage products which contain the virus-
derived sequence. With the aid of the helicase Cas3, the
mature CRISPR RNAs function as small guide RNAs that
enable cascade to inhibit virus proliferation. This finding
supports the hypothesis first put forward by Makarova and
colleagues that some transcribed CRISPRs are degraded into
small RNAs that correspond to each spacer with half of the
repeated region on either end (Tang et al., 2002, 2005) and
these small RNAs serve as the basis for an RNA interference
(RNAi) mechanism that is directed at genes expressed by
phages resulting in resistance to phage predation (Makarova
et al., 2006).
Based on their findings with E.coli K12, and in conjunction
with the available research to date on CRISPRs, Brouns and
colleagues concluded that the transcription of CRISPR
regions and the cleavage of pre-CRISPR RNA (pre-crRNA)
into mature crRNAs by Cas proteins is the molecular basis of
the CRISPR/Cas system which effectively enables
prokaryotes to combat phage attack. (Brouns et al., 2008)
The proposed mechanism of action of CRISPRs is
summarised below with the aid of figures 4a and 4b.

Figure 4a. See text for detailed explanation. (From Sorek et al., 2008)

Figure 4a shows CRISPRs obtaining phage derived spacers

that confer immunity. After phage attack, phage nucleic acids
proliferate in the cell and new virus particles are created
causing lysis of the majority of sensitive bacteria (Top row).
However, a small number of bacteria obtain phage-derived
spacers (Marked by an asterisk) which leads to their survival
due to CRISPR-mediated degradation of phage mRNA or
DNA.
Figure 4b. A putative, simplified model for CRISPR action. See text for detailed explanation.
(From Sorek et al., 2008)

This figure illustrates a putative, simplified model for

CRISPR action. After the spacer has been acquired from the
phage, the repeat-spacer array is transcribed into a long
precursor RNA and the repeats assume a secondary structure.
A complex of Cas proteins cleaves the CRISPR RNA
precursor at each repeat and maintains the cleavage products
that contain the virus-derived sequence. A Cas protein, a
helicase, assists mature CRISPR RNAs to serve as small
guide RNAs (sRNAs) (sRNAs contain a spacer and two half
repeats), which, when complexed with other Cas proteins,
base pair with phage nucleic acids, causing their degradation
(Brouns et al., 2008., Barrangou et al., 2007., Makarova et al.,
2006).
In summary, prokaryotes have evolved a nucleic acid based
immune system where the specificity is determined by the
CRISPR spacer sequence and the resistance is mediated by
the Cas enzymatic machinery (Brouns et al., 2008).

PHAGE RESPONSE TO PROKARYOTIC

CRISPR IMMUNITY
Because of the Ubiquitous nature of viruses and prokaryotes
and the parasitic interaction between them, it is not surprising
that viruses have developed mechanisms to overcome the
CRISPR immunity of prokaryotes. Deveau and colleagues
(Deveau et al., 2007) examined this topic by analysing the
sequences of a small number of phages which were still able
to infect “resistant” S.thermophilus mutants. They noted that
mutations had occurred in their sequences so that they were
no longer homologous to the spacers acquired by the
S.thermophilus mutants, thereby overcoming a central
component of the CRISPR defense. Virulent phages are
continuously rapidly evolving through nucleotide mutations
and deletions in response to the selective pressure imposed by
CRISPR and this may partly explain the very high
evolutionary rates observed in phages (Deveau et al., 2007,
Sorek et al., 2008).

THE IMPLICATIONS FOR PHAGE

POPULATION DYNAMICS AND MICROBIAL
COMMUNITIES
A recent study by Andersson and colleagues (Andersson et
al., 2008) indicates that community stability is achieved by
rapid but compensatory shifts in the levels of resistance in the
host and virus population structure. Any increase in the
immunity level of a microbial population by the acquisition of
spacers is effectively countered by phage mutation (Deveau et
al., 2007), recombination and possibly some other as of yet
uncharacterised methods of genome evolution (Peng et al.,
2004). A phage could become so virulent that it causes a crash
in the host population or resistance of the microbial
population may gradually increase to the point that the phage
population declines. However a relatively stable phage and
microbial community results when CRISPR and viral
evolution are balanced (Andersson et al., 2008).

APPLICATIONS OF CRISPR
Since its discovery, some of the following applications of the
CRISPR system have been proposed:

The engineering of resistance in industrially important

bacteria.
Because many important industries are dependent on bacteria,
such as the dairy industry, significant culture losses due to
phage infection is costly, and the dairy industry in particular
invests heavily in attempts to fight phage infection of dairy
bacteria, such as Lactobacillus.

Figure 5. Transformation of an engineered CRISPR locus into sensitive bacteria, conferring

immunity to the bacteria (From Sorek et al., 2008).

CRISPRs may be used to increase the immunity of starter

cultures by artificially inserting spacers that are taken from
the conserved regions of known phages, into the CRISPR
locus and this CRISPR system is then transformed into the
relevant bacteria, giving immunity (Horvath et al., 2008).
Spoligotyping - a CRISPR based strain typing method.
Spoligotyping is a method of strain typing which was
developed based on the CRISPR system, even though its
development pre-dated the characterisation of the CRISPR
system. It is used to detect and differentiate strains of bacteria.
Figure 6 below illustrates the methodology.
Figure 6. Spoligotyping. See text for detailed explanation (From Sorek et al., 2008)

CRISPR is amplified using the labelled primers (a and b)

which are designed from the repeat region, Probes which
match known spacers are printed on a membrane and the
amplified products for each of the isolates are then hybridised.
The black boxes indicates the presence of a spacer and the
white boxes indicate the absence of a spacer. In this example
(Fig.6), it can be seen that isolates 1 and 3 belong to the same
strain, and isolates 2, 6, and 7 belong to the same strain.
Selective silencing of endogenous genes as an alternative to
knockout methods.
Knocking out a gene or genes by traditional methods is labour
intensive, however the same effect may be achieved by
transforming a CRISPR-containing plasmid into the organism
in question, with only one of the spacers being changed to
match the gene to be studied. This occurs without
manipulation of the original microbial genome, a major
advantage of this method. Figure 7 below illustrates this
method.

Figure 7. Silencing of endogenous genes. See text for detailed explanation (From Sorek et
al., 2008)

Fragments from the chromosome (green) – encoded gene are

engineered into a CRISPR array as spacers, leading to the
silencing of the endogenous gene. This area of study has the
potential to revolutionise the field of microbial genetics and
microbial-physiology research (Sorek et al., 2008).

CONCLUSION
Prokaryotes show remarkable diversity and metabolic
capabilities which is why they grow and flourish in almost
every ecosystem where forms of life have been discovered
(Emond et al., 2007). Bacteriophages are also ubiquitous in
these same ecosystems, and also in other environments, such
as in commercially important industrial settings. Since phages
infect prokaryotes, it is not surprising that prokaryotes have
developed a number of defense mechanisms to combat phage
predation. The subject of this review is the most recently
characterised defense system, CRISPRs, which in conjunction
with Cas genes confer prokaryotes with resistance to phages.
This CRISPR system is undoubtedly different to the other four
known antiphage mechanisms, namely abortive infection,
adsorption inhibition, DNA ejection inhibition and restriction-
modification systems (Deveau et al., 2007). The CRISPR
system is very broad and effective showing wide- ranging
efficacy against phages which is in agreement with the
research which has shown that CRISPRs are found in a large
number of bacterial genomes which have been sequenced
(Godde and Bickerton., 2006., Horvath et al., 2007, Makarova
et al., 2006). The key discovery in the function of CRISPRs to
date is that the specificity of resistance is determined by the
CRISPR spacer sequence and the actual resistance is mediated
by the Cas enzymatic machinery (Brouns et al., 2008).
Although such discoveries are encouraging, there still remains
some uncharacterised elements of CRISPR structure and
function, such as the unknown functions of most Cas proteins,
highlighting the need for continuing research of this
interesting topic.
CRISPR loci do not grow unchecked (Tyson and Buffield.,
2007) and it is important to consider the counter-response of
phages to this prokaryotic immune system (Deveau et al.,
2007) in the context of homeostasis in phage and microbial
communities (Andersson et al., 2008).
To conclude, this represents a new and exciting area of
research which has the potential to lead to major
breakthroughs in the field of microbial genetics and microbial
physiology research as well as increasing our understanding
of phage-microbe interactions in ecosystems and other
environments.

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ACKNOWLEDGEMENTS
I would like to thank my literature review supervisor, Dr. Douwe
van Sinderen, for his sound advice during the compilation of this
review.
Marcas O Muineachain, December 2008.