The Future of Agriculture:Grand Challenges and Technological

Change

Moscow, March 3. 2016 National Research University Higher School of Economics

Molecular mutagenesis by genome editing

Ervin Balázs

MTA ATK Center for Agricultural Research

H-2462 Martonvásár, Hungary

Since more and more primary structure of the genetic material

of different organisms has been described, our understanding

on the different genomes rapidly evolved.

The first transgenic organism constructed by Paul Berg more

than four decades ago, and the revolution of the molecular

biology made it possible to extend that technology for almost

all living organisms.

In the case of higher plants the first transgenic tobaccos were

produced in the same time in two independent laboratories in

the US lead by Mary Dell Chilton and in Belgium headed by

Marc Van Montague and Jeff Schell.

These outstanding achievements attracted almost all

molecular biology laboratories all over the world, and

successively more and more transgenic organisms including

plants have been reported.

In the case of plants the first targets were the molecular use of

biological control of plant diseases, and included the

production of herbicide tolerant crops.

The series of insect resistant, herbicide tolerant and virus

resistant plants commercial production started in 1996.

Today their cultivation exceeded yearly 180 thousands of

hectares in the world.

The introduction of these crops is in the forefront of the debate

among different stakeholders, and almost completely blocked

in the European Union countries.

The major concerns of the opponents based on that fact that

the genetic material contains foreign genes originating from

other organisms which new combinations may not be formed

in the nature.

However the latest new methods of genome editing made it

possible that even a single nucleotide addition from different

organism not produced by these techniques.

These four molecular scissors are either directed by DNA,

RNA or Proteins all just produced mutations on already

existing genes in the organisms, just activating the silent gene

of the organisms or blocking them.

It is also possible with these techniques that a useful traits

form the same species can be incorporate into a commercial

varieties.

These mutants cannot be distinguished from naturally evolving

mutants.

These four techniques the oligo nucleotide directed

mutagenesis, the zinc finger nucleases, the TALE nucleases

and the CRISPR/Cas9 systems are efficient technologies

Specific gene editing methods

CRISPR/Cas9

oligonucleotides

Oligonucleotide Targeted Nucleotide Exchange (OTNE)

• successful generation of herbicide tolerant

variants of different crop species.

Review by Breyer et al. (2009)

Major limitations:

• low frequency

• Identification of the non-selectable mutations

Advantages:

• Not requiring transgene introduction

• Simple design

• Cheap synthesisG

G

A

T

CG

Corrected sequence

AT

mGFP

Oligo

mGFP GFP

mutant

Non-functional

GFP gene

corrected

GFP gene

OTNE

Oligonucleotide Targeted Nucleotide

Exchange (OTNE)

Correcting oligonucleotide:

5’-CCACC ATG GTG AGC AAG GGC GAG GAG CTG TTC ACC GGG

GTG-3’

(Dong et al., 2006)

Generation of mGFP transgenic maize

cell lines

ubi ubimGFP PAT

• Biolistic delivery

• PPT selection

• Sequencing

• mGFP transgenic maize cell lines are non-fluorescent!

Construction of vector:

mGFP-transgenic

maize cells

(non-fluorescent)

delivery of GFP-correcting

oligos via particle

bombardment

GFP expressing

fluorescent cells

fluorescence

microscopy

flow cytometry

Monitoring the nucleotide exchange

events

The frequency of GFP positive cells is used as an indicator of gene repair efficiency

Fluorescence stereo and confocal microscopy imaging of GFP positive cells

cell division

Restoration of GFP function

a

fluor. stereo microscope

Gene correction efficiency:

15-35 GFP positive/million cells

Quantification of GFP positive cells by

flow cytometer

144

15

315

35

182

30

0

50

100

150

200

250

300

350

GFP SDO GFP SDO GFP SDO

Nu

mb

er o

f G

FP p

osi

tive

ce

ll/1

06

even

ts

Rep.1 Rep.2 Rep.3

wtGFP oligo wtGFP oligo wtGFP oligo

Targeted genemodification

Genomic DNA

Donor DNA

Guide RNA

Cas9

complementer genomesequence

Gene therapy

Transgenic animals

cells

repair

• Negative regulator of muscle growing

• Inhibiting myoblast terminal differentiation and proliferation

• Protect myoblasts from apoptosis

• Lack of the protein causes hyperplasia/hypertrophia

• Fatty acid composition can be altered

Cloning

Molecular work

In vitro transcription

• Streptococcus pyogenes Cas9 (SpCas9)

• NLS signal

• Purified mRNA from Sigma (500 ng/ul, Sigma Ald.)

• Concentration

• 150 ng/ul

• 55 ng/ul

• No 5' UTR exon

• 7th Chromosome in rabbit

• 5 target was designed

X X XX X

Possible off targets

78

In genes

0

1 2 3 4 5

X

+

Acknowledgements

Thanks are due to

Dow AgroSciences, (Exact)

Professor Dénes Dudits (ODM)

Dr László Hiripi CRISPR/Cas9

for providing slides to this lecture