Bacterial Immunity: The

CRISPR/Cas System
Guest Lecture
Shannon Brent, Sam Mihalco, Jared
Hanninen

Outline
1.
2.
3.
4.
5.

Introduction to CRISPR/Cas System

The CRISPR Loci
Mechanism of CRISPR/Cas Immunity
Evolution of CRISPR/Cas System
Applications

What does CRISPR/Cas Stand For?

Clustered Regularly Interspaced Short
Palindromic Repeats
&
CRISPR Associated Proteins

CRISPR: clustered regularly

interspaced short palindromic repeats
Only in prokaryotes (bacteria and archaea)
Defense system: immunological memory
Relies on non-translated RNAs

CRISPR Loci

Repeat Sequences and Spacer

Sequences
Repeat sequences: palindromic, conserved
within a single loci, variation between loci
Spacer sequences: highly variable within a
single loci
Repeat

Spacer 1

Repeat

* = highly variable sequences

Spacer 2

Repeat

Spacer 3

Repeat

Spacer 4

Repeat

Leader Sequence
Contains promoter

Leader

Repeat

Spacer 1

Repeat

Spacer 2

Repeat

Spacer 3

Repeat

Spacer 4

CRISPR array = Leader sequence + Repeat sequences + Spacer sequences

Repeat

Cas genes

Intergenic
Region

Cascade

Recap: CRISPR Loci

Repeat Sequences: conserved within a loci,
flanking variable sequences
Spacer Sequences: variable, acquired from
phages
Leader Sequence: initiates transcription of the
array
Cas genes
Cascade: endonuclease activity
Cas 3: endonuclease and helicase activity
Cas 1 : endonuclease activity

Outline
1.
2.
3.
4.
5.

Introduction to CRISPR/Cas System

The CRISPR Loci
Mechanism of CRISPR/Cas Immunity
Evolution of CRISPR/Cas System
Applications

Three Steps of CRISPR/Cas Immunity

1. CRISPR Adaptation
2. CRISPR Expression
3. CRISPR Interference

1. ADAPTATION

HOST CELL

Invading Bacteriophage

Leader
Sequence

S
S
S
R
R
R
1
2
3

1. ADAPTATION
PAM = proto-spacer
adjacent motifs
Recognition site for
bacterial Cas proteins.

HOST CELL

Foreign DNA

Invading Bacteriophage

PAM

Leader
Sequence

S
S
S
R
R
R
1
2
3

1. ADAPTATION
Cas1 is a protein with
endonuclease activity.
Cuts an 80bp fragment of
foreign DNA.

Invading Bacteriophage

Cas1

HOST CELL

Foreign DNA

PAM

Leader
Sequence

S
S
S
R
R
R
1
2
3

1. ADAPTATION
80bp fragment is called a
Proto-Spacer
Invading Bacteriophage

Cas1

HOST CELL

Foreign DNA

PAM

ProtoSpacer

Leader
Sequence

S
S
S
R
R
R
1
2
3

1. ADAPTATION
The Proto-Spacer is
integrated into the CRISPR
Array, closest to the Leader
Sequence.

Invading Bacteriophage

Cas1

HOST CELL

Foreign DNA

PAM

ProtoSpacer

Leader
Sequence

S
S
S
S
R
R
R
R
3
0
1
2

1. ADAPTATION

HOST CELL

The new spacer (80bp of foregin DNA) has been integrated

and is ready to be transcribed into RNA.
Having the spacer integrated into the genome prepares the
cell to launch an immune response to that target DNA.

S
Leader
S
S
S
R
R
R
R
R
3
Sequence
0
1
2

Three Steps of CRISPR/Cas Immunity

1. CRISPR Adaptation
2. CRISPR Expression
3. CRISPR Interference

2. EXPRESSION

HOST CELL

ADAPTATION: The new spacer (80bp of foregin DNA) has

been integrated and is ready to be transcribed into RNA.
The Leader Sequence controls the transcription of the
CRISPR Array as well as Cas Proteins.

S
Leader
S
S
S
R
R
R
R
R
3
Sequence
0
1
2

2. EXPRESSION
Transcription of the array produces a single transcript called
Pre-crRNA

HOST CELL

Transcription of CRISPR Array

Pre-crRNA

S
Leader
S
S
S
R
R
R
R
R
3
Sequence
0
1
2

2. EXPRESSION
The Cascade Complex cleaves Pre-crRNA at repeat sites to
form mature crRNA

HOST CELL

Cascade Complex (CasA-E Proteins)

Pre-crRNA transcript

S
Leader
S
S
S
R
R
R
R
R
3
Sequence
0
1
2

2. EXPRESSION

HOST CELL

The Cascade Complex cleaves Pre-crRNA at repeat sites to

form mature crRNA

Cleavage by Cascade Complex

S
Leader
S
S
S
R
R
R
R
R
3
Sequence
0
1
2

2. EXPRESSION
The Cascade Complex cleaves Pre-crRNA at repeat sites to
form mature crRNA

HOST CELL

Mature crRNA

S
Leader
S
S
S
R
R
R
R
R
3
Sequence
0
1
2

Mature crRNA

Repeats

Spacer Sequence (Targets

Foreign Viral DNA)

Cascade
Complex (Cas
Proteins A-E)

2. EXPRESSION
Mature crRNAs + Cascade Complex are now
ready to perform RNA-Mediated DNAinterference.

HOST CELL

Mature crRNAs

S
Leader
S
S
S
R
R
R
R
R
3
Sequence
0
1
2

Three Steps of CRISPR/Cas Immunity

1. CRISPR Adaptation
2. CRISPR Expression
3. CRISPR Interference

3. INTERFERENCE

HOST CELL

The crRNA will specifically

bind to the complimentary
target sequence on the viral
DNA.

New Infection

Viral DNA
(Targeted)
crRNA Target Sequence
S
Leader
S
S
S
R
R
R
R
R
3
Sequence
0
1
2

3. INTERFERENCE

HOST CELL

New Infection

Viral DNA
(Targeted)

S
Leader
S
S
S
R
R
R
R
R
3
Sequence
0
1
2

3. INTERFERENCE

HOST CELL

The degradation of foreign

DNA effectively inactivates
the phage.

Inactivated Phage

Phage DNA Degradation

S
Leader
S
S
S
R
R
R
R
R
3
Sequence
0
1
2

Recap: Mechanism of Immunity

The bacterial cell must integrate foreign DNA
into its own genome before it can launch an
immune response.
Upon new infection, the pre-crRNA transcript
will include complimentary sequences to
facilitate the degradation of invading viral
DNA.

How was CRISPR system acquired?

Horizontal gene transfer

Stability
Evolutionarily unstable in the sense that is
plastic
Heritable = vertical transmission

Recap: CRISPR Evolution

CRISPR evolved through horizontal gene
transfer of foreign DNA that eventually lead to
the favourable combination of the CRISPR
genes
Constant integration and removal of CRISPR
spacers
Older spacers are furthest away from the
leader sequence

Applications
-CRISPR becoming widely utilized tool set
-Fields including
Genetics
Genomics
Agriculture
Food Science

http://us.123rf.com/

CRISPR/Cas-9 early application

-Used to modify mouse genome
-Useful Nicking enzyme
-Incorporate fluorescently/radioactively labelled
nucleotides for DNA studies

http://www.hudsonalpha.org/sites/default/files/mouse_0.jpg

Cong, Le et al. 2013. Multiplex Genome Engineering using CRISPR/Cas Systems. Science.

Capitalizing off of CRISPR

-Biotech companies on board
-System Biosciences has CRISPR/Cas-9 products
-Assist in genomic modification processes

-gene knockouts
-targetted genome editing

http://www.darkdaily.com/wp-content/uploads/524.jpg

Systembio.com 2013. Cas-9 SmartNuclease System

Human Applications
-Cas proteins studied for use in human therapies
-Shows promise as HIV-1 antiviral and potential for
actual cure
-targetting provirus promoter regions
-mutated and exised viral genome from host cells

http://cdn.zmescience.com/wp-content/uploads/2011/02/HIV-Virus.jpg

Hirotaka E. et al. 2013. Harnessing the CRISPR/Cas9 System to Disrupt Latent HIV-1 Provirus. Scientific Reports.

At the Industrial Level

-Implications for Dairy industry
-CRISPR modifications of dairy cultures shows promise
-Bacteriophage immunity through engineered CRISPR
loci
-heritable immunity
-quality control
-big savings
-less waste

http://farm3.static.flickr.com/2418/1542491708_c784185149.jpg

Mills S. et al. 2009 CRISPR analysis of bacteriophage-insensitive mutants (BIMs) of industrial Streptococcus thermophilus implications for
starter design. Journal of Applied Microbiology.

Agricultural Applications
-CRISPR/Cas technology edging into plant genomics
-Genome editing and targeted mutation
GMO production
Crop improvement
Genomic studies

http://www.scielo.cl/fbpe/img/ejb/v4n3/3/bip/f1.jpg

Xie K. et al. 2013. RNA Guided Genome editing in Plants Using a CRISPR-Cas System. Molecular Plant.

Summary
The CRISPR/Cas system is the closest thing
bacteria have to an immune system.
Uses RNA-mediated DNA-degradation via
three steps
Adaptation, Expression, Interference

Summary
The genomic content of the CRISPR/Cas
System is heritable.
The system is evolutionarily unstable, due to
its plasticity.
There are several important applications of
the CRISPR/Cas system
Industrial, agricultural, genome editing, etc.

Thank you!
Any Questions?