ABSTRACT
BRINER, ALEXANDRA ELIZABETH. CRISPR-Cas Systems in Lactic Acid Bacteria. (Under the direction of Dr. Rodolphe Barrangou).
Clustered regularly interspaced short palindromic repeats (CRISPR) and associated
proteins (Cas) are a DNA-encoded, RNA-guided, DNA-targeting adaptive immune system in
bacteria and archaea that provide protection against foreign DNA sequences from phages,
plasmids, and other mobile genetic elements. 213 Lactobacillus genomes were investigated,
and 146 CRISPR-Cas loci were identified in 131 genomes (64.9% of genomes), which is
higher than the general rate of occurrence of CRISPR-Cas systems in bacteria (~45%).
CRISPR-Cas systems are also prevalent in bifidobacteria genomes and were identified in 37
of 48 genomes examined (77%). Throughout the course of these studies, 113 diverse Type II
CRISPR-Cas systems were identified that include all necessary interference machinery,
namely the Cas9 protein, CRISPR repeats, and tracrRNA. Additionally, Protospacer
adjacent motifs (PAMs) were predicted for 14 novel systems, of which two were shown to be
active and capable of targeting plasmids with protospacer sequences identical to native
CRISPR array spacers flanked by the predicted PAM sequence. Through investigation of
these Type II systems in lactobacilli and bifidobacteria, conserved sequence and structural
modules were identified within the crRNA:tracrRNA guide duplex that are necessary for
Cas9 activity. Beyond adaptive immunity, other applications of CRISPR-Cas systems in
lactobacilli and bifidobacteria were investigated, including the genetic typing of
© Copyright 2015 Alexandra Elizabeth Briner
CRISPR-Cas Systems in Lactic Acid Bacteria
by
Alexandra Elizabeth Briner
A thesis submitted to the Graduate Faculty of North Carolina State University
in partial fulfillment of the requirements for the degree of
Master of Science
Food Science
Raleigh, North Carolina
2015
APPROVED BY:
_______________________________ ______________________________
Rodolphe Barrangou, Ph.D. Todd R. Klaenhammer, Ph.D.
Committee Chair Biotechnology Minor Chair
BIOGRAPHY
Alexandra Briner has always known she was going to be a scientist. Born in
Cleveland, OH, she would spend hours on end reading about dinosaurs or playing with her
first microscope given to her by her grandfather Briner on her eighth birthday. After 10 long
years in the land of snow, Allie, her parents, and three younger sisters packed up the family
van and moved to the great state of Georgia. It was there that she realized her love of college
football. In Athens, Allie was introduced to food science. Without hesitation, Allie fully
embraced all that food science had to offer including studying abroad in Zurich, Switzerland
and participating in the Food Science Club.
In the summer of 2013, Allie moved to Raleigh, North Carolina to begin a Master’s
degree under the advisement of Dr. Rodolphe Barrangou in the Department of Food,
Bioprocessing, and Nutrition Science at North Carolina State University. While at NCSU,
Allie continued her participation in Food Science Club, taking an active role as Treasurer and
two-time Dairy Bar Co-Chair at the NCSU State Fair. Allie also minored in Biotechnology
while pursuing an M.S. in Food Science. Upon completion of her Master’s, Allie plans to
ACKNOWLEDGEMENTS
I would like to thank all of the people who supported my learning and development while at
NCSU.
Dr. Rodolphe Barrangou for investing in me as his first-ever graduate student. I am very honored to be able to always hold that nostalgic “First Graduate Student” title, in
what is sure to become a lab with a very prolific legacy.
Dr. Todd Klaenhammer for allowing me to use his lab space and supplies while the
Barrangou lab lacked a permanent home. Thank you for sharing your extensive
wealth of information and your passion for science.
Dr. Suzanne Johanningsmeier for serving on my committee and reminding me to
think about the application of Food Science in my research.
Dr. Chase Beisel for being an extra pair of CRISPR-focused eyes willing to give
advice and guidance whenever necessary.
Dr. Emily Henriksen for everything. Thank you for refining my techniques and
continually answering all of the countless questions I ask you every day.
Rosemary Dawes, Dr. Sarah O’Flaherty, Dr. Jun Goh, Evelyn Durmaz, Brant
Johnson, Kurt Selle, Emmy Stout, Kat Daughtry, and Jeff Hymes for supporting me
with any random questions I had and allowing me to co-habit with them for two
TABLE OF CONTENTS
LIST OF TABLES………..vi
LIST OF FIGURES………...vii
LIST OF ABBREVIATIONS………ix
CHAPTER 1: BIOLOGY, GENETICS, AND APPLICATIONS OF CRISPR-CAS SYSTEMS 1 1.1INTRODUCTIONTOCRISPR-CASSYSTEMS ... 2
1.2CRISPR-CASBIOLOGYANDGENETICS ... 5
1.2.1 Acquistion... 5
1.2.2 Expression ... 8
1.2.3 Interference ... 10
1.3APPLICATIONSOFNATIVEANDENGINEEREDCRISPR-CASSYSTEMS ... 14
1.3.1 Exploiting native CRISPR systems ... 15
1.3.2 Uses of native or engineered CRISPR-Cas systems ... 19
1.3.3 Applications of engineered CRISPR-Cas systems ... 23
1.4CONCLUSION ... 25
1.5REFERENCES ... 26
CHAPTER 2: LACTOBACILLUS BUCHNERI GENOTYPING ON THE BASIS OF CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEAT (CRISPR) LOCUS DIVERSITY ... 46
2.1CONTRIBUTIONTOTHEWORK ... 47
2.2ABSTRACT ... 48
2.3INTRODUCTION ... 49
2.4MATERIALSANDMETHODS ... 52
2.4.1 Bacterial strains, media and growth conditions. ... 52
2.4.2 In silico analyses ... 52
2.4.3 DNA sequencing of L. buchneri CRISPR-Cas systems ... 54
2.4.4 Sequence deposit ... 55
2.5RESULTS ... 56
2.5.1 Identification and characterization of CRISPR-Cas systems in L. buchneri genomes. ... 56
2.5.2 Diversity of Type II-A CRISPR loci. ... 57
2.5.3 Origin of CRISPR spacers and locus activity ... 58
2.6DISCUSSION ... 61
2.7ACKNOWLEDGEMENTS ... 64
2.8REFERENCES ... 65
3.3INTRODUCTION ... 81
3.4RESULTS ... 82
3.4.1 Identification of sgRNA functional modules ... 82
3.4.2 Conservation of modules in Type II-A CRISPR-Cas systems ... 86
3.4.3 Crossing Cas9:sgRNA orthogonality boundaries ... 87
3.5DISCUSSION ... 89
3.6EXPERIMENTALPROCEDURES ... 91
3.6.1 sgRNA engineering and DNA cleavage ... 91
3.6.2 Orthogonal sgRNA:Cas9 system engineering... 92
3.7AUTHORCONTRIBUTIONS ... 93
3.8SUPPLEMENTALINFORMATION ... 93
3.9ACKNOWLEDGEMENTS ... 94
3.10REFERENCES ... 95
CHAPTER 4: OCCURRENCE AND DIVERSITY OF CRISPR-CAS SYSTEMS IN THE GENUS BIFIDOBACTERIUM ... 102
4.1AUTHORCONTRIBUTIONTOTHEWORK ... 103
4.2ABSTRACT ... 104
4.3INTRODUCTION ... 105
4.4MATERIALSANDMETHODS ... 108
4.4.1 In silico Analyses ... 108
4.4.2 RNASeq Analyses ... 109
4.5RESULTS ... 110
4.5.1 Occurrence and diversity of CRISPR-Cas systems in Bifidobacterium ... 110
4.5.2 CRISPR spacer homology to prophage sequences ... 113
4.5.3 Transcriptomic characterization of CRISPR-Cas elements ... 115
4.6DISCUSSION ... 117
4.7ACKNOWLEDGEMENTS ... 119
4.8REFERENCES ... 120
CHAPTER 5: CONCLUSIONS AND PERSPECTIVES ... 134
5.1CONCLUSIONSANDPERSPECTIVE ... 135
APPENDICES ... 136
APPENDIXA ... 137
APPENDIXB ... 146
APPENDIXC ... 148
LIST OF TABLES
Table 1.1 Cas9 proteins characterized to date ...37
Table 1.2 Uses of native and engineered CRISPR-Cas systems ...38
Table 2.1 Lactobacillus buchneri strains used in this study ...70
Table 2.2 L. buchneri CRISPR spacer matches ...72
Table 4.1 Occurrence of CRISPR-Cas systems in Bifidobacterium ...124
Table D.1 Oligos used for plasmid construction ...169
LIST OF FIGURES
Figure 1.1 CRISPR-Cas locus architecture ...39
Figure 1.2 Novel CRISPR acquisition ...40
Figure 1.3 crRNA biogenesis during the expression stage ...41
Figure 1.4 Targeting and cleavage during the interference stage ...42
Figure 1.5 Cas9 interference complex ...43
Figure 1.6 CRISPR-based genotyping ...44
Figure 1.7 Developing phage resistance through iterative phage challenges and CRISPR adaptation ...45
Figure 2.1 Type II-A CRISPR-Cas systems ...73
Figure 2.2 PCR-based detection of the CRISPR-Cas elements in L. buchneri isolates ...74
Figure 2.3 CRISPR spacer overview ...75
Figure 2.4 PAMs ...76
Figure 2.5 Sequence and structural details for core CRISPR-Cas system elements ...77
Figure 3.1 sgRNA Functional Modules ...98
Figure 3.2 crRNA:tracrRNA Module Conservation ...100
Figure 3.3 The Nexus and Hairpins Dictate Orthogonality between sgRNAs and Cas9 ...101
Figure 4.1 Clustering of Cas1 into distinct phylogenetic groups...128
Figure 4.2 CRISPR-Cas locus architecture ...129
Figure 4.3 CRISPR repeat-spacer array size distribution ...130
Figure 4.5 crRNA:tracrRNA duplex binds with target DNA sequence next to PAM
sequence ...132
Figure D.1 Maximum likelihood phylogeny derived from 73 core genes across 213 strains with CRISPR annotations ...173
Figure D.2 Comparative analysis of CRISPR sequences ...175
Figure D.3 Cas9 and tracrRNA diversity in lactobacilli ...176
Figure D.4 Cas9 diversity in lactic acid bacteria ...177
Figure D.5 Diversity of tracrRNA:crRNA duplexes and PAM sequences ...178
LIST OF ABBREVIATIONS
Clustered regularly interspaced short palindromic repeats ... CRISPR
CRISPR associated complex for antiviral defense ... Cascade
CRISPR-associated sequences ...Cas
CRISPR ribonucleoproteins ... crRNP
CRISPR RNA ... crRNA
Precursor CRISPR RNA ...pre-crRNA
Protospacer adjacent motif ... PAM
Single guide RNA ...sgRNA
CHAPTER 1: BIOLOGY, GENETICS, AND APPLICATIONS
1.1 INTRODUCTION TO CRISPR-CAS SYSTEMS
All organisms have a need to protect themselves against foreign invaders. Since the early
2000s, researchers have been studying the ability of CRISPR-Cas adaptive immune systems
to protect bacteria and archaea against potentially damaging exogenic nucleic acids and
mobile genetic elements (MGEs) (Jansen 2002, Bolotin 2005, Haft 2005, Mojica 2005,
Pourcel 2005, Godde 2006, Makarova 2006, Barrangou 2007). Clustered regularly
interspaced short palindromic repeats (CRISPR) and associated genes (cas) work as the
immunization records and effector molecules for bacterial immunity against phages,
plasmids, and potentially harmful nucleic acids through a three stage process of acquisition of new spacer sequences derived from foreign DNA, expression and biogenesis of small CRISPR RNAs (crRNAs) that guide Cas proteins in the interference stage to destroy invaders that the cell has stored immunity against (Figure 1) (Barrangou 2007, Terns 2011,
Barrangou and Marraffini 2014, Fineran 2014, van der Oost 2014, Westra 2014). These
immune systems are estimated to occur in less than 50% of bacteria and in over 80% of
archaea (as of the 8-2014 update of CRISPRdb) (Grissa 2007). All CRISPR-Cas systems
have repeat-spacer arrays with conserved repeats between 24 and 47 nucleotides flanking
variable spacer sequences that are derived from the MGEs, as well as CRISPR
ribonucleoprotein (crRNP) complexes that are responsible for recognizing and cleaving
invading foreign nucleic acids (Kunin 2007, Barrangou 2007, Makarova 2011, Makarova
2013, van der Oost 2014). All CRISPR systems contain Cas proteins that group into four
functional categories: i. Proteins with nuclease and recombinase activities for acquisition and
Ribonucleases for processing CRISPR RNAs (crRNAs); iii. Proteins that assemble into
crRNP complexes that include Cascade, Cas9, Cmr, and Csm; and iv. Nucleases for
degradation of foreign MGEs include Cas3 and Cas9 (Wiedenheft 2009, Bhaya 2011, Yosef
2012, Makarova 2013, Barrangou and Marraffini 2014, Carte2014, Jackson 2014, van der
Oost 2014, Westra 2014).
There are three types of CRISPR-Cas systems that are distinguished by repeat length
and sequence, gene content, crRNA biogenesis, and effector protein-mediated target
destruction (Figure 1) (Makarova 2011, Koonin 2013, Makarova 2013). All CRISPR-Cas
systems contain the universal cas1 gene, but each system type as a unique signature gene that
can help identify them: cas3, cas9, and cas10, respectively for Types I, II and III (Wiedenheft 2009, Makarova 2011). Type I and Type III systems have been found in
bacteria and archaea, and, while phylogenetically different, are very similar in the structure
and function of crRNPs in the three stages of CRISPR immunity; Type III systems are
primarily found in archaea (Bhaya 2011, Terns and Terns 2013, van der Oost 2014). Type I
systems employ a large multi-protein complex called Cascade (CRISPR-associated complex
for antiviral defense) and Type IIIA and IIIB systems employ a csm or cmr complex, respectively, to target and cleave MGEs when guided by a crRNA (Makarova 2011,
Barrangou 2013, Makarova 2013, van der Oost 2014). All systems target double stranded
DNA except Type IIIB systems which have been shown to target RNA (Hale 2009, Terns
and content of cas genes but always contain cas3, cas1, and cas2 (Makarova 2011). Recently, a new subtype of Type Is, Type I-U, was identified that contains a novel cas gene layout and content Each subtype has a slightly different assemblage of Cas proteins in the
Cascade complex. Type II systems contain three or four cas genes and perform foreign DNA
targeting and cleavage through only one protein, Cas9, and an additional RNA molecule (the
tracrRNA) (Gasiunas 2012, Chylinski 2013). Type III immunity is not as extensively
characterized as the other systems and requires further insights into immune functions.
Type II CRISPR-Cas systems are very different than their Type I and III counterparts.
Type II systems are very rare, having only been found in 5% of bacteria and not found in any
archaeal genomes (Chylinski 2014). While these systems are rare in nature, human
commensal organisms (like lactic acid bacteria and bifidobacteria) and some human
pathogens have an unusually high rate of occurrence for Type II systems (Horvath 2009,
Fonfara 2014, Chylinski 2014). Horizontal and vertical gene transfer of Type II systems
amongst organisms occupying similar niches may support the high rate of occurrence in
human-associated organisms (Takeuchi 2012). Type II CRISPR-Cas systems are not
restricted to solely human-associated organisms, however, and have been found in diverse
environments, including salt and fresh water, soil, fermentation environments, and extreme
temperature conditions like hot springs and Antarctic ice (Fonfara 2014, Takeuchi 2012).
There are three subtypes of Type II systems, namely II-A, II-B and II-C; while all Type II
systems employ an additional RNA molecule, the trans-activating CRISPR RNA (tracrRNA),
systems contain cas9, cas1, and cas2 genes; Type II-A and II-B systems contain an additional csn2 or cas4 gene, respectively, that is hypothesized to assist Cas1 during the acquisition stage (Barrangou 2007, Makarova 2011, Makarova 2013).
1.2 CRISPR-CAS BIOLOGY AND GENETICS
CRISPR-Cas systems provide immune protection to the cell in three steps: 1.) acquisition
of a nucleic acid sequence from an invader, 2.) expression and biogenesis of small interfering
RNA units, and 3.) interference and cleavage of foreign nucleic acids by crRNPs upon re-introduction into the cell (van der Oost 2009, Bhaya 2011, Barrangou and Marraffini 2014,
Westra 2014). The three stages of CRISPR immunity vary by the effector proteins and
interference molecules depending on the type of CRISPR-Cas system.
1.2.1 Acquistion
Native acquisition of immunity against phages has only been seen in one organism,
Streptococcus thermophilus (Barrangou 2007); however, several studies have been able to achieve “primed” acquisition of spacers showing homology to previously acquired spacers or
to invaders that have already been identified and bound by Cascade, which was typically
achieved through heterologous expression of cas1 and cas2 genes (Li 2014, Pul 2010, Pougach 2010, Datsenko 2012, Yosef 2012, Swarts 2012, Erdmann 2012, Kiro 2013, Westra
phages are bound to Cas proteins, new spacers are acquired from the intruder and CRISPR
immunity is restored (Datsenko 2012, Fineran 2014).
Recent studies have revealed the structure and mechanism for Cas1- and
Cas2-mediated incorporation of new DNA spacers into the repeat arrays (Arslan 2014, Nunez
2014), showing both proteins are required for recognition of the leader region and integration
of new spacers; the leader region is an AT-rich transcriptional promoter adjacent to the first
repeat (Wei 2015). Cas1 acts as an integrase and causes a staggered cut across the
leader-proximal repeat; the new double stranded spacer DNA is ligated between the two single
stranded repeats (Figure 2) (Arslan 2014). In Type IE systems, incorporation of a new spacer
is initiated when the CRISPR repeat forms a cruciform structure. First the hydroxyl group on
the 3’ guanine nucleotide of the minus strand if the repeat is attacked by the double stranded
spacer sequence; this forms a half sit intermediate where the new spacer is bound to the 3’
end of the first repeat. A second nucleophilic attack occurs on the hydroxyl group at the other
end of the repeat on the on the plus strand. This second attack creates an integrated spacer
flanked by single stranded DNA repeats. After DNA repair to complement the repeats, the
new spacer is fully incorporated into the array (Figure 2) (Nunez 2015). While the function
of Cas2 is not completely known, Cas1 cannot function properly without the formation of a
Cas1-Cas2 complex, thus preventing new spacer acquisition (Nunez 2014). In some systems,
Cas4 may be necessary for helicase activity and Csn2 may be involved in DNA stability or
recruitment of other cellular machinery. The nuclease activity of Cas1 is necessary to cleave
the repeats during acquisition. Conversely, inactivation of the nuclease activity of Cas2
added at the leader-proximal end, the order in which spacers are added provides a
chronological history of immunization events (Andersson 2008, Tyson 2008).
Acquisition occurs when the Cas proteins identify a foreign DNA sequence, called a
protospacer, that is flanked on one edge by a protospacer-adjacent motif (PAM)
approximately 2 to 4 nucleotides long (Deveau 2008, Mojica 2009, Shah 2013). When
stored in the repeat-spacer array, a spacer sequence is not adjacent to a PAM, therefore
preventing the CRISPR-Cas systems from self-targeting and cleaving the host chromosome
(Marraffini 2010, Stern 2010, Swarts 2012). The sequence stored in the repeat-spacer array is
referred to as the spacer, while the homologous sequence on the foreign DNA is referred to
as the protospacer (Deveau 2008). In Type I systems, the PAM sequence is generally located
on the 5’ end of the protospacer, while the PAM in Type II systems is located on the 3’ flank
(Deveau 2008). Recent data on acquisition in Type II systems more fully demonstrated that
Cas9 in complex with Cas1, Cas2, and Csn2 binds the PAM sequence on a potential
protospacer to ensure only functional targets are stored in the repeat-spacer array (Heler
2015). Additionally, the nuclease activity of Cas9 is not necessary for Type II acquisition
(Heler 2015). Type III systems are hypothesized to be PAM-independent, utilizing an
unknown mechanism for acquisition; self versus non-self discrimination during interference
may be dependent on an absence of complementarity between the target strand and the
1.2.2 Expression
After the acquisition stage, the entire repeat-spacer array is transcribed into a single
RNA transcript that contains all the repeats and spacers encoded in the locus (Figure 3)
(Bhaya 2011). In order to become efficacious, the pre-crRNA must be cleaved into smaller
crRNA molecules that contain a partial CRISPR repeat and partial spacer (Brouns 2008).
The repeats in Type I systems are palindromic and form hairpin structures (Kunin 2007);
Cas6 cleaves the base of the repeat-hairpins forming small interfering crRNAs with an entire
spacer sequence flanked by eight nucleotides of the CRISPR repeat on the 5’ end and
followed by a repeat-hairpin on the 3’ end (Figure 3) (Carte 2008, Carte 2010, Carte 2014,
Hochstrasser 2015, Li 2015). Type I-C CRISPR-Cas systems are the only system to utilize
Cas5 as an endoribonuclease to cleave the pre-crRNA strand (Hochstrasser 2015, Li 2015).
The expression stage of Type III systems is not fully understood, but Cas6 and the Csm/Cmr
complex is still likely to be involved (Hale 2005, Carte 2014).
Type II CRISPR-Cas systems utilize an additional RNA molecule, the tracrRNA, to
generate crRNAs (Figure 3 and Figure 4). The tracrRNA contains a partial complementary
anti-repeat at the 5’ end that allows the molecule to base pair with the repeat portion of the
crRNA (Deltcheva 2011, Gasiunas 2012, Chylinksi 2013, Karvelis 2013, Chylinski 2014,
Briner 2014). This complementary region forms three structural modules that are conserved
in Type II-A tracrRNA:crRNA duplexes and are important for molecular stability and
binding ability with Cas9: the upperstem, bulge, and lowerstem (Briner 2014). The CRISPR
repeat often contains a G as the first nucleotide of the repeat that forms a G-U base wobble at
comprised of several hair pin structures that are key in nucleotide binding interactions with
Cas9 (Jinek 2012, Jinek 2013, Nishimasu 2013, Anders 2014, Briner 2014). The first hairpin
structure in the tracrRNA, called the nexus, can take several structural forms, but often has a
conserved nucleotide sequence in the base of the hairpin stem (Briner 2014). The
streptococci nexus structures contain a single hairpin with an off-set uracil nucleotide; the
nexus in Streptococcus pyogenes and Streptococcus thermophilus CRISPR3 is three base-pairs long while the Streptococcus thermophilus CRISPR1 nexus is four base-pairs long (Briner 2014). Lactobacilli usually contain a double-stemmed hairpin with a symmetrical,
round bulge in the middle of the hairpin. While the structure of the nexus is only conserved
within genera, the sequence UNANNC beginning at the G-U wobble at the lower stem is
highly conserved amongst all Streptococcus and Lactobacillus tracrRNA sequences (Briner 2014). Downstream of the nexus, the terminal hairpins vary in number, size, and structure but
usually contain a Rho-independent transcriptional terminator hairpin that is GC rich and
followed by a string of uracils at the 3’ end (Deltcheva 2011, Chylinski 2013, Karvelis 2013,
Chylinski 2014, Briner 2014). The hairpins and nexus are key factors in determining Cas9
orthogonality and cross-compatibility (Esvelt 2013, Briner 2014, Fonfara 2014).
During the expression stage of CRISPR-Cas immunity in Type II systems, the
pre-crRNA containing all of the repeat and spacer sequences is transcribed into one molecule; the
anti-repeat portion of the tracrRNA is able to base pair with the repeat portions of the
(Deltcheva 2011, Karvelis 2013). A secondary unknown mechanism then trims the crRNA
at the 5’ end of the spacer portion of the crRNA so that only 19 to 20 nucleotides of a partial
CRISPR spacer are left intact (Karvelis 2013). In minimal repeat-spacer arrays containing a
single spacer flanked by repeats, no RNase III processing is necessary and the
crRNA:tracrRNA duplex retains both flanking repeats during the interference stage (Karvelis
2013). The crRNA:tracrRNA duplex remains bound to Cas9 during the interference stage
(Jinek 2012).
1.2.3 Interference
Once crRNAs have been generated, they will work to guide the crRNP to their
complementary protospacer target where the Cas proteins and other endogenous nucleases
will be able to target, cleave, and degrade the invading nucleic acid during CRISPR
interference. Although Type I and Type III systems contain vastly different cas genes, the crRNP proteins for Cascade and the Cmr/Csm complexes are structurally very similar,
comprised of multi-protein subunits that form a sea-horse shaped complex that is able to bind
both the guide RNA sequence and the target MGE (Bhaya 2011, Jore 2011, Terns and Terns
2013, van der Oost 2014). Once the Cascade complex in Type I systems has bound to its
target foreign sequence, a conformational change of the complex triggers the recruitment of
Cas3, a 3’ to 5’ nuclease and helicase, to unwind and degrade the foreign DNA (Brouns
2008, Gong 2014, Jackson 2014).
When foreign DNA enters a cell with a Type II CRISPR-Cas system, Cas9 first binds
between the spacer and protospacer (Figure 4) (Sternberg 2014). If the 3’ end of the spacer
sequence does not show perfect homology to the spacer sequence, Cas9 will not cleave the
DNA (Deveau 2008, Wiedenheft 2011, Gasiunas 2012, Chen 2014, Sternberg 2014).
Additionally, if the PAM is not present, Cas9 will not bind the target thus preventing
cleavage (Sapranauskas 2011, Chen 2014).
Cas9 is a bi-lobed protein containing an alpha-helical recognition (REC) lobe and
nuclease (NUC) lobe (Jinek 2012, Chylinksi 2013, Nishimasu 2013, Anders 2014, Fonfara
2014). There are several conserved motifs found in all Cas9s that include: an HNH nickase,
three RuvC domains, and an arginine-rich bridge (Gasiunas 2012). Within the HNH and
RuvC domains, there are several catalytic residues that are necessary for cleavage activity
(Sapranauskas 2011, Gasiunas 2012, Fonfara 2014). While the length of Cas9 varies
between Type II subtypes, these domains can be detected in all Cas9 proteins. Type II-C
Cas9s tend to be the shortest in length, ranging from ~900 to ~1100 amino acids, while some
of the largest II-A Cas9s, over 1,300 amino acids. Interestingly, II-A systems can be further
classified into II-A systems with long Cas9s (1,350-1,389 amino acids) and II-As with short
Cas9s that are closer in length to some II-C Cas9s (Chylinski 2013, Chylinski 2014).
Recent crystal structures of the Streptococcus pyogenes and Actinomyces naslundii Cas9s reveal major conformational changes occur between the unbound state,
guideRNA-bound state, and guideRNA:target DNA guideRNA-bound state (Jinek 2012, Jinek 2013, Nishimasu
attachment (Figure 4). When the S. pyogenes guide RNA is loaded into the Cas9, a positively charged grove forms between the REC and NUC lobes and provides a channel for
the target DNA to bind (Jinek 2013, Nishimasu 2013, Anders 2014). Once the guide RNA
and target DNA are complexed with Cas9, the protein will begin to search for and bind to a
PAM sequence. Upon binding to the PAM, an R-loop structure is formed in which the guide
RNA hybridizes with one strand of the target DNA and displaces the other DNA strand
(Jinek 2013, Nishimasu 2013). The strand opposite of the PAM is interrogated for
complementarity with the spacer sequence on the guide. Interrogation proceeds in the 3’ to
5’ direction through the spacer starting at the seed sequence; the seed sequence is the first
eight to 10 nucleotides of the spacer and is imperative to have complete complementary in
this region as mismatches will prevent Cas9 cleavage (Semenova 2011, Wiedenheft 2011,
Jiang 2013, Kunne 2014, Sternberg 2014). The RNA:DNA hybridization and creation of an
R-loop structure is thought to be the final trigger needed for Cas9 cleavage. The HNH
domain nicks the strand paired with the crRNA, and the RuvC domain nicks the displaced
strand containing the PAM; cleavage occurs exactly three nucleotides from the 3’ edge of the
spacer (Jinek 2013, Nishimasu 2013, Sternberg 2014). The Streptococcus pyogenes Cas9 appears to be a single-turnover, blunt, double-stranded nickase endonuclease. While
1.2.3.1 tracrRNA modules are important for Cas9 functionality
The tracrRNA has been recognized as an extremely important molecule in allowing
the Cas9:guide RNA:target DNA complex to form. Several nucleotides and structural
elements in the tracrRNA are necessary to enable Cas9 binding and cleavage (Jinek 2013,
Nishimasu 2013, Anders 2014). The binding of the crRNA and tracrRNA form five
functional modules found in all II-A guide RNA structures: the upper stem, bulge, lower
stem, nexus and hairpins (Briner 2014). Three modules (the upper stem, bulge, and lower
stem) are directly formed by the repeat-anti-repeat complementarity, and the nexus and
hairpin modules are formed by the tracrRNA secondary structures (Deltcheva 2011, Gasiunas
2012, Chylinski 2013, Chylinski 2014). Based on the Streptococcus pyogenes Cas9 guideRNA- and target DNA-bound structure, the phosphate backbone of the upper and lower
stem, formed by the repeat-anti-repeat binding, interacts in a sequence-independent manner
with Cas9, demonstrating the crRNA repeat structure is more important than the repeat
sequence (Nishimasu 2013, Anders 2014). Located just after the stem region of guide RNA,
the nexus hairpin is a conserved structure of varying size and shape; when bound to the guide
RNA and DNA target, the arginine bridge helix of the Cas9 binds with the nexus and base of
the lower stem and brings order to a region that is disordered in the apo-structure of Cas9
(Jinek 2013, Nishimasu 2013, Anders 2014).
The nexus appears to be a very integral module in the tracrRNA based on its structure
protrudes from the stem loop and binds with an asparagine residue in the REC lobe, while the
adenosine-52 residue binds with an arginine residue. Together, these interactions allow the
protein to access the double stranded DNA at the PAM site (Nishimasu 2013, Anders 2014).
The protruding U59 is conserved in all Streptococcus II-A tracrRNA sequences. Additionally, the A52 and C55 nucleotides appear to be conserved in the majority of II-A
tracrRNA sequences (Jinek 2013, Nishimasu 2013, Anders 2014). The conservation of these
nucleotides further suggests that these bases may be biochemically important for Cas9
binding interactions across all II-A systems (Briner 2014).
1.3 APPLICATIONS OF NATIVE AND ENGINEERED CRISPR-CAS SYSTEMS
The power of CRISPR-Cas systems can be harnessed for many applications outside of
natural antiviral and DNA defense mechanisms to include evolutionary studies, next
generation antibiotics, and development of transcriptional control and genetic engineering
tools (Table 2). Many industries have been able to exploit the natural immune function of
these systems to confer resistance against undesirable DNA sequences including phages,
plasmids, and antibiotic resistance genes (Marraffini 2008, Horvath 2009, Garneau 2010,
Terns 2011, Barrangou 2012). Additionally, the genetic fingerprint left behind from iterative
phage predation or plasmid introduction can leave historical timelines that can be exploited
for strain typing and detection (Andersson 2008, Horvath 2008, Barrangou 2012, Dimarzio
2013). Engineered systems can be used to harness the RNA-guided, DNA-targeting ability
of Cas9 to perform many genetic engineering tasks in prokaryotes as well as eukaryotes and
1.3.1 Exploiting native CRISPR systems
Domesticated bacteria used in dairy and bioprocessing fermentations are often plagued
by bacteriophage attack (Mahony 2015). The host-rich environment becomes a feeding
playground for phages that thrive under industrial fermentation settings. In order to combat
constant phage-predation, lactic acid bacteria (LAB) and bifidobacteria have developed
numerous phage-defense systems that include: inhibiting absorption and DNA injection,
restriction-modification systems (R-M systems), abortive infection, and CRISPR-mediated
phage defenses (Horavth 2009, Labrie 2010, Barrangou 2012, Westra 2012, Dupuis 2013,
Mahony 2015). Of these phage interference mechanisms, CRISPR immunity is the only
adaptive viral defense system that has the ability to capture resistance to specific, novel viral sequences. This adaptation to new sequences has been exploited with the dairy starter
culture Streptococcus thermophilus to produce naturally immunized strains through iterative
rounds of phage infection (Figure 6) (Barrangou 2007, Horvath 2008, Barrangou 2013). By
exploiting the natural immune capabilities of CRISPR-Cas systems, dairy and
biofermentation processors can produce phage-resistant starter cultures with relative ease and
reduce loss caused by tanks failing due to lytic phage infection (Mahony 2015, Barrangou
ARFST). In 2007, Barrangou and colleagues were the first to definitively correlate the
genotype of spacer sequences with the phenotype of phage resistance. Using S. thermophilus
CRISPR1, a Type II system, they demonstrated when spacers matching a phage were
phage was added to the CRISPR array. They were able to define the bacteriophage
insensitive mutant (BIM) phenotype as CRISPR-spacer encoded immunity (Barrangou
2007).
The fingerprint left by CRISPR-Cas acquisition and immunity in bacterial and
archaeal genomes provides a genetic locus that is rich with phylogenetic information. Since
the identification of CRISPR-Cas systems in the 2000s, important characteristics of CRISPR
repeats and spacer sequences have allowed CRISPR-typing to become a very powerful tool
in understanding strain divergence, ancestry, and relatedness (Andersson 2008, Tyson 2008,
Horvath 2008, Barrangou 2012).
CRISPR spacers are acquired from invading DNA from sources like plasmids and
phages and are added in a site-specific manner at the leader end of the repeat-spacer array,
meaning the newest spacers are located at the leader end of the array and the ancestral
spacers are anchored at the distal end (Figure 5) (Barrangou 2007, Tyson 2008). This
polarized acquisition provides a genetic timeline that often provide insights into the
phylogenetic relationships of very closely related strains through the conservation and
diversity seen in the hypervariable spacer region (Barrangou 2007, Tyson 2008, Horvath
2008, Barrangou 2013). When comparing two strains with the same CRISPR system,
identical ancestral spacer content is a sign of common ancestry between strains (Horvath
2008, Pourcel 2005, Tyson 2008). New, rare spacer sequences closer to the leader end of the
array are a sign of strain divergence and evolution (Tyson 2008, Horvath 2008, Deveau
2008). While variation of spacer sequences in the leader-end of the array can usually be
nucleotide polymorphisms (SNPs) in CRISPR-repeats can also be a sign of strain divergence
through loss of spacer sequences by homologous recombination (Figure 5) (Horvath 2008,
Barrangou 2007, Tyson 2008, Fang 1998, Levin 2013).
Identifying strain relatedness through CRISPR spacer sequences has been used as a
new tool for strain typing and detection in the fields of epidemiology and ecology. CRISPR
repeat-spacer arrays from many strains of a single organism can be sequenced and compared
to reveal phylogenetic relationships like strain divergence and ancestral relatedness
(Barrangou 2012). This kind of typing has been extensively studied in the food-borne
pathogen Salmonella as well as in the yogurt manufacturing organism, Streptococcus thermophilus (Anderson 2008, Tyson 2008, Horvath 2008, DiMarzio 2013, Fabre 2012, Liu 2011a, Liu 2011b). Out of the 2,500 Salmonella enterica subsp enterica serovars, over 1,000
of them have been analyzed using two of the known CRISPR loci to exist in this species
(DiMarzio 2013, Fabre 2012, Liu 2011a, Liu 2011b). Using solely CRISPR loci, a clear
correlation has been shown to exist between CRISPR locus content of Typhimurium serovar
isolates and antibiotic resistance genes (DiMarzio 2013). The epidemiological insights
gained from using CRISPR-based sequences can not only detect, but also simultaneously
subtype Salmonella isolates. This assay can be exploited in hospitals during outbreak situations where laboratories are trending towards implementing culture-independent
detection methods (van Belkum 2007).
adaption to phage predation is often a population-level response to selective pressure that
selects for variant strains that contain diverse spacer immune markers (Figure 6) (Tyson
2008, Andersson 2008); because CRISPR adaptation can happen rapidly in environments
with phage predation, the evolution at CRISPR loci occurs much faster in a population than
nucleotide polymorphism accumulation (Tyson 2008). Through the process of phage attack
and host acquisition of immunity, phages and host cells co-evolve; phages evolve through
single nucleotide mutations and deletions to avoid sequence recognition by host CRISPR
machinery, while the host cell acquires new spacer sequences (Hiedelberg 2009, Deveau
2008, Datsenko 2012, Barrangou 2013). Additionally, the ability of CRISPR-Cas systems to
influence HGT has been documented as a naturally occurring process that affects bacterial
genome evolution (Bikard 2012, Palmer and Gilmore 2010, Hatoum-Aslan 2014). In
Enterococci, there is a correlation between the absence of CRISPR-cas systems and the accumulation of antibiotic resistance genes; this suggests CRISPR-Cas systems take part in
preventing natural HGT in this genus (Palmer and Gilmore 2010). The ability to target phage
and plasmid DNA and widely prevent HGT, suggests CRISPR-Cas systems play an
important role in shaping bacterial evolution (Barrangou 2007, Garnaeu 2010, Marraffini
2008, Palmer 2010, Bikard 2012).
Through identification of the protospacer origins, CRISPR loci can provide further
insights into environmental microbial and phage ecology (Bolotin 2005, Pourcel 2005). In
metagenomic datasets, CRISPR spacers have been used to reverse engineer phage genomes
as well as identify bacterial hosts from virome studies (Andersson 2008, Stern 2012,
CRISPR repeat-spacer arrays to assemble and identify phage sequences in metagenomic data
(Stern 2012). They were able to identify 991 phages and 130 bacterial hosts from several
individuals gut microbiome. Through the identification of the origin of the CRISPR spacers
and host containing the CRISPR repeats, a picture of a rich host-phage environment emerged
from the human gut microbiome (Stern 2012). In a similar manner, a study by Anderson and
colleagues of the marine virome yielded little insights of viral diversity due to lack of viral
sequences in publically available databases (Anderson 2011). Viral sequences were then
compared for similarity to a database of all known CRISPR spacers; this comparison
identified homology between viruses and CRISPR spacers allowing researchers to identify
the host organism for these viruses. Many viral sequences were uncategorized before
identification through CRISPR spacer homology due to a lack of known viral sequences in
databases (Anderson 2011). Because CRISPR-Cas systems record sequences from MGEs,
the repeat spacer sequences of these loci can reveal information about the environmental and
ecological state of phage-host interplay.
1.3.2 Uses of native or engineered CRISPR-Cas systems
Much recent CRISPR-Cas research has focused on the ability to repurpose or redirect
Cas machinery to act as a DNA binding or cleaving tool for bacterial strain enhancement or
genetic engineering (Selle and Barrangou 2015, Bikard 2013, Luo 2014, Bikard 2012).
sequences, native systems can be exploited to guide Cas machinery to desired DNA
sequences for binding or cleavage of the target. Heterologous expression of engineered
CRISPR-Cas systems in non-native bacteria can also be used to perform the same genetic
engineering tasks that native CRISPR-Cas systems can perform (Sapranauskas 2011, Bikard
2012, Bikard 2013, Bikard 2014, Selle and Barrangou 2015).
CRISPR-Cas systems are able to target plasmid DNA in addition to phage DNA
(Garneau 2010, Terns and Terns 2011 Curr Opin Microbiol, Marraffini 2008). This ability
has been exploited to design strains with active CRISPR-Cas immune systems that are able to
prevent plasmid uptake by incorporating a CRISPR spacer into an array with sequence
identity to a plasmid sequence (Marraffini 2008 Science, Garneau 2010). Additionally,
non-native CRISPR systems can be introduced heterologously into a bacteria and provide
effective CRISPR immunity (Bikard 2012). The blocking of plasmid uptake and
dissemination can help prevent the horizontal transfer of undesirable genes, like antibiotic
resistance genes, that are often harbored on plasmids (Marraffini 2008 Science, Wiedenheft
2012 Nature, Bikard 2012 Cell Host Microbe). CRISPR-Cas systems that are engineered to
prevent the of uptake of foreign DNA can be programmed to limit HGT and prevent
emergence of virulence factors, like antibiotic resistance and capsule genes, that allow
bacteria to adapt to harsh environments (Marraffini 2008, Bikard 2012).
Artificial and native CRISPR-Cas systems can be engineered to produce
sequence-specific antimicrobials that can be used to alter or remove native populations of bacteria.
Targeting chromosomal DNA using CRISPR-based crRNPs has been shown to be a
Bikard 2014). In order to survive this cyotoxic event, CRISPR-Cas systems that target the
host chromosome are inactivated through deletion or mutations of the cas genes, deletion of
the self-targeting spacer from the repeat-spacer array, or deletion or mutation of the target
site in the chromosome; these mutations can cause major genome reshaping events that lead
to genome evolution (Vercoe 2013). Two main studies by Gomaa et al., and Bikard et al.,
demonstrated the ability to engineer CRISPR-Cas elements to target and cleave the
chromosome of specific bacteria. Gomaa et al, relied on naturally occurring Type I-E
systems in Escherichia coli. They transformed a plasmid containing a spacer sequence that
was unique to one genome into a mixed population two different strains of E. coli. They were able to see the destruction of only the E. coli that contained the protospacer sequence (Gomaa 2013). Bikard et al., used phage delivery of Cas9 and a guide RNA to target and
remove undesirable microbes in a mixed population (Bikard 2014). They were able to target
only kanamycin-resistant Staphylococcus aureus, effectively removing only members of the population with the gene of interest. This targeted removal shown by Gomaa et al. and
Bikard et al. has the potential to be harnessed to develop next generation smart antibiotics
with the ability to selectively remove only undesirable bacteria based on specific genetic
sequences (Gomaa 2013, Bikard 2014).
Through deactivation of the nuclease activities of Cas9 and Cas3 in Type II and I
systems, respectively, researchers have been able to develop powerful DNA-targeting
machinery. In bacteria with Type I systems, once the cas3 gene has been deleted or inactivated, transcriptional repression has been shown to be very effective when artificial
guides are designed to target gene promoter regions (Luo 2015). Type II systems require that
the nickase resides at D10 and H840 in the Streptococcus pyogenes Cas9 be deactivated in order to abolish the nuclease function of Cas9 (Bikard 2013). Once the residues have been
deactivated (D10A and H840A), Cas9 can be used in the native cell or transferred to a
non-native background for transcriptional control repurposing. Fusion of Cas9 to the omega
subunit of an RNA Polymerase was shown to act as a transcriptional activator in bacterial
genomes (Bikard 2013). Studying transcriptional control in bacteria allows researchers to
better understand bacterial gene function and cellular pathways.
Beyond transcriptional control, active Cas proteins have been used for genome
editing and genetic engineering in native and non-native bacterial systems. Through
provision of a guide RNA, Cas proteins are directed to a sequence-specific location in the
chromosome where a cleavage event occurs. While self-targeting of CRISPR-Cas systems is
shown to be a cyto-toxic event, repair mechanisms present in the cell can fix double stranded
breaks caused by Cas nucleases and lead to incorporations of mutations, insertions, and
deletions in the host chromosome (Selle and Barrangou 2015). Large deletions as a result of
Cas self-targeting results in genome simplification and can be of great interest for
evolutionary biologist as well as the bioprocessing industries interested in utilizing efficient,
streamlined bacteria as their industrial work horses (Selle unpublished). Genetic engineering
of desired strains is an important skill to utilize when generating bacteria for bioprocessing,
proteins for genetic engineering and genome editing will greatly aid the ease and efficiency
of altering and enhancing bacterial genetics.
1.3.3 Applications of engineered CRISPR-Cas systems
Through the understanding of native CRISPR-Cas functionality in bacterial cells, the
interference property of Cas9 guided by a crRNA:tracrRNA duplex has been implemented as
a genetic engineering tool with endless potential (Doudna and Charpentier 2014). The recent
boom in applications of CRISPR-based technology for genome editing and genetic
engineering has been coined the “CRISPR Craze” (Table 2) (Pennisi 2013). The main draw
to CRISPR-Cas9 tools is the ability to program Cas9 to bind and cleave a specific DNA
sequence through the alteration of the guide RNA that is a chimeric molecule that mimics the
crRNA:tracrRNA duplex (Jinek 2012, Cong 2013, Jinek 2013, Mali 2013). Artificial guide
RNAs can be used to replace the crRNA:tracrRNA duplex by artificially joining the 3’ end of
the crRNA to the 5’ end of the tracrRNA by a four-nucleotide linker (Jinek 2012); this
improvement reduced the four component system (Cas9, tracrRNA, repeat-spacer arrays, and
RNase III) to a two component system (Cas9 and guide RNA) and improved the ease and
efficiency of using CRISPR-Cas9 based tools for genetic engineering in eukaryotic
backgrounds. Through deactivation of the catalytic residues within the HNH and RuvC
nicking domains, Cas9 can be turned into a protein that simply targets and does not cleave
Cas9 cleavage causes double stranded breaks (DSBs) exactly three nucleotides away
from the PAM sequence on the target DNA. Researchers can harness repair machinery
native to cells to aid the cell in recovery from DSBs and incorporate insertions, deletions, and
mutations in genes of interest to study disease models, analyze gene function, correct genetic
mutations, and generate animal and cell line models (Doudna 2014). Non-homologous
end-joining (NHEJ) creates small insertions and deletions at the DSB site; homology directed
repair (HDR) allows for whole gene correction or insertion of a large sequence through the
addition of a template that is used for assisted recombination. NHEJ and HDR are not the
only means for DNA repair in a cell, but they are currently the most frequently used for
Cas9-mediated DSB recovery (Doudna 2014). When using a dCas9, no DSB occurs. Instead,
Cas9 can be used as a DNA-targeting tool and can be fused with other proteins or molecules
for tasks like protein recruitment, transcription activation and repression, and fluorescent
tagging for visualization of genomic regions, chromatin remodeling, and methylation and
epigenetics (Doudna 2014, Tanenbaum 2014, Chen 2014). Cas9 can also be multiplexed to
allow for several genetic engineering events to occur simultaneously in a single cell (Cong
2013). Cas9-based genome engineering has been used to engineer human cell lines, model
animal organisms, plants, fungi, and bacteria across many scientific fields (Zhou 2014,
Doudna 2014). Harnessing the potential uses for Cas9 has just begun and is expected to
1.4 CONCLUSION
CRISPR-Cas systems are adaptive immune systems in bacteria and archaea that utilize Cas
proteins directed by RNA to target and degrade DNA from foreign genetic invaders.
CRISPR immunity occurs in three stages: i. acquisition of new spacer sequences by Cas1 and
Cas2 from MGEs that invade cells, ii. expression and biogenesis of individual crRNAs that contain a partial CRISPR repeat and partial spacer matching foreign DNA, iii. interference and cleavage of foreign DNA upon reintroduction into the cell by Cas machinery and a
crRNA guide. All CRISPR immunity occurs in these stages but the Cas proteins, crRNA
biogenesis, and interference mechanism is different for the three different types of
CRISPR-Cas systems. Elements from Type II CRISPR-CRISPR-Cas systems, including the CRISPR-Cas9 protein,
tracrRNA, and crRNA, have been used to develop genetic engineering tools in bacteria and
eukaryotes. Beyond genetic engineering, native CRISPR immunity has been used to confer
resistance against undesirable DNA sequences including phages, plasmids, and antibiotic
resistance genes (Marraffini 2008, Horvath 2009, Garneau 2010, Terns 2011, Barrangou
2012). Additionally, the genetic information gained from iterative phage predation or
plasmid introduction can leave historical timelines that can be exploited for strain typing and
detection as well as provide a basis for studying bacterial and phage evolution and ecology
(Andersson 2008, Horvath 2008, Barrangou 2012, Dimarzio 2013). CRISPR-Cas systems of
archaea and bacteria hold powerful potential to be utilized and exploited in many realms of
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