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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

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© Copyright 2015 Alexandra Elizabeth Briner

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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

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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

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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

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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

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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

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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

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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

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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

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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

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CHAPTER 1: BIOLOGY, GENETICS, AND APPLICATIONS

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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

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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

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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),

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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

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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

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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

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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

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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

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(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

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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

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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

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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

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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

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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

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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

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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).

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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,

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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).

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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

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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

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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,

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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

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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

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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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Figure

Table 1.1 | Cas9 proteins characterized to date
Table 1.2 | Uses of native and engineered CRISPR-Cas systems
Figure 1.2 | Novel CRISPR acquisition
Figure 1.6 | CRISPR-based genotyping
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References

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