Inhoud
Les 01: Applications of CRISPR/Cas genome editing in microbiology.................................................. 2
1. The CRISPR revolution .......................................................................................................... 2
1.1 Biological origins of CRISPR ................................................................................................. 2
2. CRISPR in prokaryotic immunity ............................................................................................ 3
2.1 Stage 01 – Adaptation or spacer acquisition ......................................................................... 3
2.2 Stage 02 – crRNA biogenesis ................................................................................................ 4
2.3 Stage 03 – Target interference .............................................................................................. 4
2.4 CRISPR-Cas9 (Type II-A) ...................................................................................................... 5
3. CRISPR/Cas9 is a tool for genome engineering ...................................................................... 7
3.1 CRISPR-mediated genome editing ....................................................................................... 8
3.2 CRISPR/Cas variants ........................................................................................................... 9
3.3 CRISPR-mediated genome editing in practice: the toolbox .................................................. 10
3.4 CRISPR-mediated genome editing: applications ................................................................. 11
3.5 CRISPR-Cas9: selected applications.................................................................................. 11
3.6 Viral host factor identification ............................................................................................ 13
4. Coronaviruses .................................................................................................................... 13
4.1 Identifying viral host factors via genome-wide CRISPR screens............................................ 14
4.2 What is the role of TMEM106B in SARS-CoV-2 infection? ..................................................... 15
4.3 Conclusions...................................................................................................................... 17
1
, Les 01: Applications of CRISPR/Cas genome editing in microbiology
1. The CRISPR revolution
CRISPR is becoming more important in the broader course context, as the number of publications on the topic is
steadily increasing.
CRISPR’s development began with fundamental
research driven by scientific curiosity → how
does this work, without any knowledge
80’ systems how they could target enzymes +
zinc fingers. Proteins that bind to specific DNA
sequence. But for genome editing, the challenge
is to target a very specific locus in the genome.
CRISPR biology can be applied to genome
editing.
Protein engineering was required to make these
proteins bind to THAT specific locus.
Fundamental research can be a big step forward!
1.1 Biological origins of CRISPR
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) was first discovered as a part of the
natural immune system in bacteria. These microorganisms use CRISPR-associated proteins (Cas) to defend
themselves against viruses (bacteriophages).
• When a virus infects a bacterium, the bacterium
can “capture” a small piece of the viral DNA and
insert it into its own genome within the CRISPR
region.
• These viral DNA fragments serve as a “memory”
of past infections.
• If the same virus attacks again, the bacterium
uses the stored sequences to produce CRISPR
RNAs (crRNAs), which guide Cas proteins (like
Cas9) to recognize and cut the viral DNA,
effectively neutralizing the threat.
The most widely used version is CRISPR-Cas9, where:
• A guide RNA (gRNA) is designed to match a specific DNA sequence.
• The Cas9 enzyme cuts the DNA at that exact location.
• This allows researchers to either disable a gene, insert new genetic material, or correct mutations.
2
, 2. CRISPR in prokaryotic immunity
The CRISPR-Ca system is a type of adaptive immune system found in bacteria and archaea. It protects them
form invading viruses (called bacteriophages) by recognizing, remembering, and then destroying viral genetic
material.
2.1 Stage 01 – Adaptation or spacer acquisition
When a virus infects a bacterium for the first
time, the bacterium captures a small piece of
the virus’s DNA and stores it in its own genome.
This stored DNA acts as a “memory” of the
virus.
• The CRISPR region in the bacterial
genome contains repeated DNA
sequences, separated by spacers
(unique DNA segments).
- Spacer: 20 NT-vreemd DNA → wordt geïntegreerd in het genoom van de bacterie
o Spacer zal niet altijd 20 NT lang zijn, dit kan verschillen.
• When a virus infect the bacterium, special Cas proteins (Cas 1 & Cas2) cut a small piece of the viral
DNA.
- Cas 1 en Cas 2 detecteert het DNA en knipt een klein stukje → integreert dat in het genoom van
de bacterie.
• This piece is inserted into the CRISPR array as a new spacer.
• This means the bacterium now has a genetic “memory” of that virus, so it can recognize it next time.
! A spacer is a short piece of DNA in the CRISPR array that comes from a virus (bacteriophage) that previously
infected the bacterium. It serves as a genetic memory of past infections.
! Cas stands for CRISPR-associated proteins. These are enzymes (like Cas9) that perform specific functions,
such as:
• Cutting DNA (they are endonucleases: enzymes that cut inside DNA strands).
• Grabbing and processing RNA
• Binding to target DNA based on guide RNA instructions
Phage infection: protospacer (a sequence of the invading DNA) is incorporated into the CRISPR array by specific
Cas proteins.
CRISPR-Cas system:
• CRISPR array: identical repeats interspersed by phage-derived spacers
- Verschillende spacers met daartussen de repeats
• Cas operon, usually in vicinity (= Cas-operan bevindt zich meestal in de buurt van het CRISPR-assay in
het genoom van de bacterie)
3
Les 01: Applications of CRISPR/Cas genome editing in microbiology.................................................. 2
1. The CRISPR revolution .......................................................................................................... 2
1.1 Biological origins of CRISPR ................................................................................................. 2
2. CRISPR in prokaryotic immunity ............................................................................................ 3
2.1 Stage 01 – Adaptation or spacer acquisition ......................................................................... 3
2.2 Stage 02 – crRNA biogenesis ................................................................................................ 4
2.3 Stage 03 – Target interference .............................................................................................. 4
2.4 CRISPR-Cas9 (Type II-A) ...................................................................................................... 5
3. CRISPR/Cas9 is a tool for genome engineering ...................................................................... 7
3.1 CRISPR-mediated genome editing ....................................................................................... 8
3.2 CRISPR/Cas variants ........................................................................................................... 9
3.3 CRISPR-mediated genome editing in practice: the toolbox .................................................. 10
3.4 CRISPR-mediated genome editing: applications ................................................................. 11
3.5 CRISPR-Cas9: selected applications.................................................................................. 11
3.6 Viral host factor identification ............................................................................................ 13
4. Coronaviruses .................................................................................................................... 13
4.1 Identifying viral host factors via genome-wide CRISPR screens............................................ 14
4.2 What is the role of TMEM106B in SARS-CoV-2 infection? ..................................................... 15
4.3 Conclusions...................................................................................................................... 17
1
, Les 01: Applications of CRISPR/Cas genome editing in microbiology
1. The CRISPR revolution
CRISPR is becoming more important in the broader course context, as the number of publications on the topic is
steadily increasing.
CRISPR’s development began with fundamental
research driven by scientific curiosity → how
does this work, without any knowledge
80’ systems how they could target enzymes +
zinc fingers. Proteins that bind to specific DNA
sequence. But for genome editing, the challenge
is to target a very specific locus in the genome.
CRISPR biology can be applied to genome
editing.
Protein engineering was required to make these
proteins bind to THAT specific locus.
Fundamental research can be a big step forward!
1.1 Biological origins of CRISPR
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) was first discovered as a part of the
natural immune system in bacteria. These microorganisms use CRISPR-associated proteins (Cas) to defend
themselves against viruses (bacteriophages).
• When a virus infects a bacterium, the bacterium
can “capture” a small piece of the viral DNA and
insert it into its own genome within the CRISPR
region.
• These viral DNA fragments serve as a “memory”
of past infections.
• If the same virus attacks again, the bacterium
uses the stored sequences to produce CRISPR
RNAs (crRNAs), which guide Cas proteins (like
Cas9) to recognize and cut the viral DNA,
effectively neutralizing the threat.
The most widely used version is CRISPR-Cas9, where:
• A guide RNA (gRNA) is designed to match a specific DNA sequence.
• The Cas9 enzyme cuts the DNA at that exact location.
• This allows researchers to either disable a gene, insert new genetic material, or correct mutations.
2
, 2. CRISPR in prokaryotic immunity
The CRISPR-Ca system is a type of adaptive immune system found in bacteria and archaea. It protects them
form invading viruses (called bacteriophages) by recognizing, remembering, and then destroying viral genetic
material.
2.1 Stage 01 – Adaptation or spacer acquisition
When a virus infects a bacterium for the first
time, the bacterium captures a small piece of
the virus’s DNA and stores it in its own genome.
This stored DNA acts as a “memory” of the
virus.
• The CRISPR region in the bacterial
genome contains repeated DNA
sequences, separated by spacers
(unique DNA segments).
- Spacer: 20 NT-vreemd DNA → wordt geïntegreerd in het genoom van de bacterie
o Spacer zal niet altijd 20 NT lang zijn, dit kan verschillen.
• When a virus infect the bacterium, special Cas proteins (Cas 1 & Cas2) cut a small piece of the viral
DNA.
- Cas 1 en Cas 2 detecteert het DNA en knipt een klein stukje → integreert dat in het genoom van
de bacterie.
• This piece is inserted into the CRISPR array as a new spacer.
• This means the bacterium now has a genetic “memory” of that virus, so it can recognize it next time.
! A spacer is a short piece of DNA in the CRISPR array that comes from a virus (bacteriophage) that previously
infected the bacterium. It serves as a genetic memory of past infections.
! Cas stands for CRISPR-associated proteins. These are enzymes (like Cas9) that perform specific functions,
such as:
• Cutting DNA (they are endonucleases: enzymes that cut inside DNA strands).
• Grabbing and processing RNA
• Binding to target DNA based on guide RNA instructions
Phage infection: protospacer (a sequence of the invading DNA) is incorporated into the CRISPR array by specific
Cas proteins.
CRISPR-Cas system:
• CRISPR array: identical repeats interspersed by phage-derived spacers
- Verschillende spacers met daartussen de repeats
• Cas operon, usually in vicinity (= Cas-operan bevindt zich meestal in de buurt van het CRISPR-assay in
het genoom van de bacterie)
3
