'CRISPR/Cas technology'

Julia Manetsberger, PhD

Laboratory of Neuronal Communication
julia.manetsberger@cme.vib-kuleuven.be
Outline
 Introduction to Genetic Engineering
 CRISPR/Cas9 technology
• Mechanism
• Variations
 Applications
• CRISPR in Research
• HIV
• Human genome editing
 Regulation
Genetic Engineering
“The deliberate modification of the characteristics of an organism by
manipulating its genetic material.”

• Research

• Medicine (Protein/Enzyme
production)

• Agriculture (Crops)

• Industrial Biotechnology
(Biofuel production)

• Entertainment
 The Way towards Genetic Engineering
Rules Information Basics Genome editing

1859 Darwin 1944 Avery– 1970 Restriction Zink Fingers

“Origin of Species” MacLeod–McCarty Enzymes
DNA as the genetic TALENs
1856-66 Mendel material 1977 Sanger
“Mendelian Sequencing CRISPRs
inheritance” 1953 Watson, Crick
and Franklin 1983 PCR
1871 Mieska DNA structure
Nucleic acids 2003 Human
1961-1967 Genetic Genome Project
code

Adapted form Doudna, 2016

Genetic Editing uses DNA Repair Pathways
Genomic DNA

Nonhomologous end-joining Homology directed repair (HDR)

(NHEJ)

Repair
template

X
Knockout Repair, Novel Function, Therapy etc.
Genome Editing using Site Specific Nucleases

Zinc Fingers

Protein based

TALEN

CRISPR
RNA based

Adapted from Duke University

 History of CRISPR
1987 1st report on repetitive sequences (CRISPR, Ishano et al.)

2000 CRISPR present throughout prokaryotes (Mojica et al.)

2005 Foreign elements, proposed immunity function (Mojica et al.)

CRISPR-Clustered Regularly Interspaced Palindromic Repeats

Adapted from Hsu, P. D et al. Cell, 2014

 History of CRISPR
1987 1st report on CRISPR (Ishano et al.)

2000 CRISPR present throughout prokaryotes (Mojica et al.)

2005 Foreign elements, proposed immunity function (Mojica et al.)

2010 Cas9 is guided by spacer and induces DSB in target (Garneau et al.)

2011 Heterologous expression of CRISPR type II (Sapranauskas et al.)

Adapted from Hsu, P. D et al. Cell, 2014

 History of CRISPR
1987 1st report on CRISPR (Ishano et al.)

2000 CRISPR present throughout prokaryotes (Mojica et al.)

2005 Foreign origin of spacers, proposed immunity function (Mojica et al)

2010 Cas9 is guided by spacer and induces DSB in target (Garneau et al.)

2011 Heterologous expression of CRISPR type II (Sapranauskas et al.)

2012 Proposal CRISPR for Genome editing (Jinek, Doudna, Charpentier et al.)

2013 CRISPR used for genome editing in eukaryotic cells (Zhang et al.)

2014 Crystal structure of Cas9 gRNA complex (Nishimasu, Zhang et al.)

Adapted from Hsu, P. D et al. Cell, 2014

Outline
 Introduction to Genetic Engineering
 CRISPR/Cas9 technology
• Mechanism
• Variations
 Applications
• CRISPR in Research
• HIV
• Human genome editing
 Regulation
Adaptive immune system

attack

defence
Adaptive immune system

Attack

Double strand
Defense Break (DSB) SURVIVAL

X
What is CRISPR?

1. Protein component

2. RNA component

3. Target component Source: Transomic

Genomic organisation of CRISPR
CRISPR-Clustered Regularly Interspaced Palindromic Repeats

CRISPR-Cas loci:

• array of identical repeats

• invader DNA-targeting spacers
• Operon of cas genes encoding Cas protein components
• Transactivating RNA

tracrRNA cas operon Repeat-spacer array

 Components and Cleavage

tracrRNA
cas operon Repeat-spacer array

Cas proteins for DNA cleavage:

Type I and Type III Type II

Components and Cleavage

• Adaptation
Recognition of target site
• Two nuclease domains
RuvC (gray) - cleaves
non-target DNA strand
HNH (cyan) - cleaves-
target strand of DNA
• PAM-interacting domain
(orange)

Cas9
(S. pyogenes)
Jinek et al. Science, 2014
 Components and Cleavage

tracrRNA
cas operon Repeat-spacer array

tracrRNA pre-crRNA

Processing

guide RNA
(gRNA)

Complex formation

Cas9:gRNA
complex
Components and Cleavage

tracrRNA
cas operon Repeat-spacer array

Target Recognition:

? ? ?
 Components and Cleavage

tracrRNA
cas operon Repeat-spacer array

Viral DNA

PAM PAM

N G G

PAM-Protospacer Adjacent Motive

Components and Cleavage

tracrRNA
cas operon Repeat-spacer array

PAM PAM
Drawback-Specificity of CRISPR/Cas9 System

Genomic DNA

CCTop - CRISPR/Cas9 target online predictor

 Enhancing specificity of CRISPR/Cas9 technology
1. Nickase Activity

Hsu, P. D et al. Cell, 2014, Jinek et al. Science, 2014

 Enhancing specificity of CRISPR/Cas9 technology
1. Nickase Activity
2. FokI-Fusion

Guilinger et al., Nature Biotechnology, 2014

 Enhancing specificity of CRISPR/Cas9 technology
1. Nickase Activity
2. FokI-Fusion
3. Improved target recognition

Slaymaker et al., Science, 2016

• Efficient • Specificity
• Versatile • Toxicity
• Easily adaptable • Delivery
(RNA level not Protein)
• Multiplexing
Adapted from Charpentier, Doudna et al., Science, 2014 and OriGene
bacteria fungi Animals/human cells plants
Adapted from Charpentier, Doudna et al., Science, 2014 and OriGene
Outline
 Introduction to Genetic Engineering
 CRISPR/Cas9 technology
• Mechanism
• Variations
 Applications
• CRISPR in Research
• HIV
• Human genome editing
 Regulation
 CRISPR to study disease genes-example LRRK
G2019

Choose locus LRRK

Cas9 gRNA

*
LRRK
gRNA, Donor design

Cas9 gRNA
Transfection of cells
with components *
LRRK

DSB generation LRRK LRRK

X * X
LRRK

Identification and characterisation G2019S

of desired mutant *
LRRK
 CRISPR/Cas9 Toolbox
1. Genome Editing

LRRK LRRK
X * X
LRRK

2. Regulating Gene Expression

Gene

3. Imaging of Genomic Loci

GFP

Gene
CRISPR and HIV

adapted from Ricochet Creative Productions LLC

CRISPR and HIV

LTR Promoter/Genes LTR

Viral DNA
CRISPR and HIV-but….

• Base substitutions
• Insertions
• Deletions
=> but ORF remains intact

NEHJ

X
CRISPR in Humane Genome Editing
 CRISPR in Humane Genome Editing

• modify the gene responsible for β-thalassaemia (potentially fatal blood

disorder)
• unviable human embryos (leftover from IVF)
• Mutation in HBB gene (human β-globin) is corrected in single cell stage
• CRISPR not efficient

*
HBB

Cyranoski & Reardon, Nature News, 2015

 CRISPR in Humane Genome Editing

• Reduce miscarriages and understand earliest

developments in life  treatment for infertility
• healthy human embryos (leftover from IVF)
• target genes (OCT4) that are active in the first few days
• Experiments will follow first 7 days
Callaway, Nature News, 2015
Outline
 Introduction to Genetic Engineering
 CRISPR/Cas9 technology
• Mechanism
• Variations
 Applications
• CRISPR in Research
• HIV
• Human genome editing
 Regulation
National Academy of Sciences (USA)
National Academy of Medicine (USA)
Chinese Academy of Sciences
The Royal Society (UK)
1. Basic and Preclinical Research.
a) Research is needed
b) modified cells should not be used to establish a pregnancy.

2. Clinical Use: Somatic.

a) Directed at altering genetic sequences only in somatic cells (sickle-cell anemia, immune cells to
target cancer).
b) Evaluation within existing and evolving regulatory framework.

3. Clinical Use: Germline.

a) relevant safety and efficacy issues have to be addressed
b) societal consensus about the appropriateness of the proposed application.
c) appropriate regulatory oversight.
d) clinical use of germline editing should be revisited on a regular basis.
4. Need for an Ongoing Forum.
a) Each nation ultimately has the authority to regulate activities under its jurisdiction, but the
human genome is shared among all nations.
b) establish norms concerning acceptable uses of human germline editing and to harmonize
regulations, in order to discourage unacceptable activities while advancing human health and
welfare.

We therefore call upon the national academies that co-hosted the summit – the U.S.
National Academy of Sciences and U.S. National Academy of Medicine; the Royal
Society; and the Chinese Academy of Sciences – to take the lead in creating an ongoing
international forum to discuss potential clinical uses of gene editing; help inform
decisions by national policymakers and others; formulate recommendations and
guidelines; and promote coordination among nations.
 polyphenol oxidase (PPO)

• Polyphenol oxidase (PPO) causes browning of mushrooms during storage

• CRISPR to introduce mutations to 1 out of 6 PPO genes
• 30% reduced activity
• Prolonged storage time

Waltz, Nature 2016

• US Department of Agriculture (USDA) will not regulate a CRISPR modified mushroom
(“No foreign DNA present”)
• Cultivated and sold without passing through the agency's regulatory process
• First CRISPR-edited organism to be approved

Waltz, Nature 2016

THANK YOU!
julia.manetsberger@cme.vib-kuleuven.be