Risk Analysis of Genome Editing: Laboratory Based Studies€¦ · analysis Determination of genome editing specificity by addressing different target regions Different approaches

Risk Analysis of Genome Editing: Laboratory Based Studies

Biological Weapons Convention - Meeting of Experts MX2

Eva Oellingrath - [email protected] - 31 July 2019, Geneva

Genome Editing

Classic genomic modifications in bacteria Induction of mutations by radiation or chemicals

First genetic modification in 1964 by Boyer & Cohen

Gene manipulation by conjugative DNA transfer

Homologous recombination

Nucleases as molecular scissors

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“Genome editing describes the specific modification of the genetic material of a living organism by deleting, replacing, or inserting a DNA sequence, typically with the aim of improving a crop or farmed

animal, or correcting a genetic disorder.” Google

BWC - Meeting of Experts MX2 Strategies for the Risk Assessment of Genome Editing

WHO article, 2019


Zinc finger Nucleases 1996

Homing Endonucleases 2011

TALEN 2009

CRISPR/CAS 2012

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Key principle of genome editing Generation of a DNA double strand

break (DSB)

Editing by Homology Directed Repair (HDR) or Non Homologous End Joining (NHEJ)

Targeted genome modification possible by homologous recombination via HDR

Tools for genome engineering

Genome Editing

BWC - Meeting of Experts MX2 Strategies for the Risk Assessment of Genome Editing 4

Key principle of genome editing Generation of a DNA double strand

break (DSB)

Editing by Homology Directed Repair (HDR) or Non Homologous End Joining (NHEJ)

Targeted genome modification possible by homologous recombination via HDR

Genome Editing

NHEJ-mediated repair HDR-mediated repair

Nuclease-induced DSB GCTCCCAACCGCTTCAAGCTCGA CTGGGTGAAGAGCCAGTTTGCTCCCA CGAGGGTTGGCGAAGTTCGAGCT GACCCACTTCTCGGTCAAACGAGGGT

3’ 5’

5’ 3’

No DNA template

Insertion Deletion

GCTCCCAATTTGCTCCCA CGAGGGTTAAACGAGGGT

3’ 5’

5’ 3’

GCTCCCAACCATTGATGGTTTGCTCCCA CGAGGGTTGGTAACTACCAAACGAGGGT

3’ 5’

5’ 3’

GCTCCCAACCGCTTC GTTTGCTCCCA CGAGGGTTGGCGAAG CAAACGAGGGT

3’ 5’

5’ 3’

+ DNA template

Gene insertion

GCTCCCAACCGCTTC CCAGTTTGCTCCCA CGAGGGTTGGCGAAG GGTCAAACGAGGGT

3’ 5’

5’ 3’


3’ 5’

5’ 3’


3’ 5’

5’ 3’

GCTCCCAACCGCTTC GTTTGCTCCCA CGAGGGTTGGCGAAG CAAACGAGGGT

3’ 5’

5’ 3’

+ DNA template

Point mutation

GCTCCCAACCATTTTGCTCCCA CGAGGGTTGGTAAAACGAGGGT

3’ 5’

5’ 3’

GCTCCCAACCATTTTGCTCCCA CGAGGGTTGGTAAAACGAGGGT

3’ 5’

5’ 3’

Possible modifications Insertion

Deletion

Point Mutation

5 BWC - Meeting of Experts MX2 Strategies for the Risk Assessment of Genome Editing

Genome Editing

CRISPR/CAS: Breakthrough of the Year 2015

Science breakthrough of the year 2015

Based on a naturally occurring mechanism

Highly precise cleavage of genetic material

High efficiency!?

Lower costs

Ability to address any selected genomic site!?

“Simple solution for solving complex problems in genome engineering”!?

Application possible in various areas: biomedicine, biotechnology, basic research, agriculture …


Science news stuff, 2015

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2005 1993 2012 2007 2002 1987 2013 2014

Yoshizumi Ishino discovered the CRISPR

sequence in E. coli

Francisco Mojica characterized the now called CRISPR

locus

Jansen and Mojica coined the term

CRISPR, discovery of signature cas gene

Alexander Bolotin identified the

biotechnology relevant Cas9 and the PAM

sequence

Horvath and colleagues first experimental demonstrated the CRISPR adaptive

immunity

Doudna and Charpentier, first idea of a synthetic guide

RNA as a new genome editing tool

Feng Zhang first demonstrated targeted

genome cleavage by Cas9 in human and mouse cells

first CRISPR patent was

granted to Feng Zheng


CRISPR/CAS: Historical Development

CRISPR/CAS – Adaptive Bacterial Immune System

Adaptive bacterial immune system against viruses and foreign DNA

Found in 90% of archaea and >50% of bacteria species

Adaptive memory by assembly of foreign fragment in bacterial genome

Elimination of foreign DNA by specific recognition and cleavage

Bacterial cell

Virus Virus

Duroux-Richard, Giovannangeli, Apparailly, 2017

RNA

RNA

RNA

RNA

RNA


Clustered Regularly Interspaced Short Palindromic Repeats/CRSIRP-associated proteins

Mohanraju et al., 2016


• Six main CRISPR/CAS types grouped in 2 classes (~45 subtypes) • All can be found in nature in bacteria and archaea!

CRISPR/CAS9: genome editing tool

Diversity of CRISPR/CAS Systems

singleguideRNA crRNA::tracrRNA::complex

CRISPR/CAS: New Genome Engineering Technology

Cas9, ThermoFisher, 2016

Cas9

Identification of target gene Design of specific sgRNA Species specific modifications Genome editing

GIFS UNITED

Highly programmable!


Target dsDNA

+

+

Bacterial cell Visual screening: identification of successfully edited bacteria

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Genome Editing with CRISPR/CAS in Bacteria


Bacteria colonies on blue/white screening plate

+ HDR Fragment

Aims of My Laboratory Studies

Basic research: • Analysis of the functional regulation of an endogenous

CRISPR/CAS system in a bacterial model organism Applied research: • Evaluation of the efficiency and specificity of CRISPR/CAS9 as

tool for genome editing in bacteria • Analysis of secondary effects in selected target organism • Assessment of the misuse potential of CRISPR/CAS tools


Basic Research: CRISPR/CAS in B. glumae

Modell organism Burkholderia glumae

Soil ß-proteobacterium

Endogenous CRISPR/CAS system

Class I multienzyme complex system

CRISPR/CAS I-F subtype

Regulation is not fully understood


Mohanraju et al., 2016

Assessment of genome editing efficiency by statistical analysis

Determination of genome editing specificity by addressing different target regions

Different approaches and analytical methods

Comparison of traditional methods vs. CRISPR/CAS tools

Investigation in different bacterial strains

Transcriptome analysis and RT-qPCR

Phenotypic characterisation

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Applied Research: CRISPR/CAS9 Tool


qPCR

Genome editing of different targets

Insertions of fluorescence marker

Deletion of metabolism marker

Evaluation by

Phenotypic characterisation

Metabolism activity assays

Whole genome sequencing

classcentral


Applied Research: CRISPR/CAS9 Tool

Possible Negative Effects of CRISPR/CAS9

On-target effects Off-target effects

Additional unspecific insertion of a targeted sequence or base pairs

Additional unspecific deletion of a targeted sequence or base pairs

Target Region Target Region

X X X X X X X X


dsDNA dsDNA

Genetic accessibility in bacteria maybe impaired by Methylation Histone-like proteins (H-NS) Secondary structure Method optimisation still required Standardised protocols Evaluation of species specific modifications Algorithms for the identification of unwanted genome edits Macmillan, 2013

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Current Limitations of Genome Editing in Bacteria


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CRISPR/CAS biology still not well characterised

On- and Off-target effects -> unwanted gene mutations

Ease of use: extensive practical & theoretical lab knowledge still required

Efficiency and specificity must be proven case-by-case

Analysis of CRISPR/CAS-based gene manipulation requires time consuming and expensive methods (e.g. whole genome sequencing)


Limitations of CRISPR/CAS as Genome Editing Tool

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Benefits of CRISPR/CAS as Genome Editing Tool


Only few components required (Cas enzyme, sgRNA, DNA template…)

High DNA cleavage precision, superior efficiency and specificity

Faster than other genome editing technologies due to lesser working steps

Programmable editing tool: delete, insert or repair genes Multiplexing allows simultaneous targeting of multiple DNA sites

CRISPR/CAS Environment Human Health

Agriculture

Human gene therapy

New genetic traits

Improve crops

Revolution or

Risk?

Biofuels

Drug development/ target ID

Bioplastics

Bioremediation

Biosensing

Pesticide reduction

Nitrogen fixation

Invasive species

Vector control: Gene drives

Kevin Esvelt, modified, 2019 Wheeler, 2006

Future Applications for Genome Editing

BWC - Meeting of Experts MX2 Strategies for the Risk Assessment of Genome Editing 20

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Misuse potential: depends on target organism, aims, capabilities

Case-by-case risk analysis required

Gene Drive technology: misuse for hostile purpose? Research on countermeasures > legitimate but problematic?

Detection of CRISPR/CAS based gene manipulation maybe difficult: attribution problems

Establishment of procedures for the responsible handling of genome editing technologies by BWC Member States


Risks of CRISPR/CAS as Genome Editing Tool

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Biological Weapons Convention - Meeting of Experts MX2

[email protected] 31 July 2019, Geneva

Thank you!

Documents

Risk Analysis of Genome Editing: Laboratory Based Studies€¦ · analysis Determination of genome editing specificity by addressing different target regions Different approaches