crispr/cas technology' - ucll · 'crispr/cas technology' julia manetsberger, phd...

'CRISPR/Cas technology'

Julia Manetsberger, PhDLaboratory of Neuronal Communication

[email protected]

Outline

Introduction to Genetic Engineering

CRISPR/Cas9 technology• Mechanism• Variations

Applications• CRISPR in Research• HIV• Human genome editing

Regulation

Genetic Engineering

• Research

• Medicine (Protein/Enzyme

production)

• Agriculture (Crops)

• Industrial Biotechnology

(Biofuel production)

• Entertainment

“The deliberate modification of the characteristics of an organism by manipulating its genetic material.”

The Way towards Genetic Engineering

Rules

1859 Darwin“Origin of Species”

1856-66 Mendel “Mendelian inheritance”

1871 MieskaNucleic acids

Information

1944 Avery–MacLeod–McCartyDNA as the genetic material

1953 Watson, Crick and FranklinDNA structure

1961-1967 Genetic code

Basics

1970 Restriction Enzymes

1977 Sanger Sequencing

1983 PCR

2003 Human Genome Project

Genome editing

Zink Fingers

TALENs

CRISPRs

Adapted form Doudna, 2016

Nonhomologous end-joining(NHEJ)

XKnockout

Homology directed repair (HDR)

Repairtemplate

Repair, Novel Function, Therapy etc.

Genetic Editing uses DNA Repair PathwaysGenomic DNA

Genome Editing using Site Specific Nucleases

Zinc Fingers

TALEN

CRISPR

Adapted from Duke University

Protein based

RNA based

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.)

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

CRISPR-Clustered Regularly Interspaced Palindromic Repeats

History of CRISPR

1987 1st report on CRISPR (Ishano 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.)


History of CRISPR

1987 1st report on CRISPR (Ishano 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.)


Outline




Regulation

attack

defence

Adaptive immune system

Adaptive immune system

Attack

SURVIVALDefense

Double strandBreak (DSB)

X

What is CRISPR?

Source: Transomic

1. Protein component

2. RNA component

3. Target component

Genomic organisation of CRISPRCRISPR-Clustered Regularly Interspaced Palindromic Repeats

CRISPR-Cas loci:

Repeat-spacer array

• array of identical repeats

• invader DNA-targeting spacers

• Operon of cas genes encoding Cas protein components

cas operon

• Transactivating RNA

tracrRNA

Components and Cleavage

Repeat-spacer arraycas operontracrRNA

Type II

Cas proteins for DNA cleavage:

Type I and Type III


Cas9(S. pyogenes)

• AdaptationRecognition of target site

• Two nuclease domains RuvC (gray) - cleaves non-target DNA strandHNH (cyan) - cleaves-target strand of DNA

• PAM-interacting domain (orange)

Jinek et al. Science, 2014


pre-crRNAtracrRNA

Processing

guide RNA(gRNA)

Complex formation

Cas9:gRNA complex




Target Recognition:

???



PAM PAM

N G G

Viral DNA

PAM-Protospacer Adjacent Motive



PAM PAM

Drawback-Specificity of CRISPR/Cas9 System

CCTop - CRISPR/Cas9 target online predictor

Genomic DNA

Enhancing specificity of CRISPR/Cas9 technology

1. Nickase Activity

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


1. Nickase Activity

Guilinger et al., Nature Biotechnology, 2014

2. FokI-Fusion


1. Nickase Activity

Slaymaker et al., Science, 2016

2. FokI-Fusion

3. Improved target recognition

• Efficient

• Versatile

• Easily adaptable (RNA level not Protein)

• Multiplexing

• Specificity

• Toxicity

• Delivery

Adapted from Charpentier, Doudna et al., Science, 2014 and OriGene

plantsbacteria fungi Animals/human cellsAdapted from Charpentier, Doudna et al., Science, 2014 and OriGene

Outline




Regulation

CRISPR to study disease genes-example LRRK

Choose locus LRRKG2019

Identification and characterisationof desired mutant

LRRK

G2019S*

Transfection of cells with components

DSB generation LRRK LRRK

LRRKXX *

LRRK

Cas9 gRNA

*

gRNA, Donor designLRRK

Cas9 gRNA

*

CRISPR/Cas9 Toolbox

1. Genome Editing

LRRK LRRK

LRRKXX *

2. Regulating Gene Expression

Gene

3. Imaging of Genomic Loci

Gene

GFP

CRISPR and HIV

adapted from Ricochet Creative Productions LLC

X

CRISPR and HIV

LTR LTR

Viral DNA

Promoter/Genes

CRISPR and HIV-but….

• Base substitutions

• Insertions

• Deletions

=> but ORF remains intact

NEHJ

X

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

Cyranoski & Reardon, Nature News, 2015

HBB*

• 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




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) 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

polyphenol oxidase (PPO)

• 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!

[email protected]

crispr/cas technology' - ucll · 'crispr/cas technology' julia manetsberger, phd...

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