Dr. Karl Ravet & Prof. Stephen Pearce

Genomics and genome editing:

New tools for developing desired traitsin CO winter wheat.

AES Research Center Conference,January 10th, 2019.

What challenges does agriculture face today?

A growing population… … in a changing environment

elevate cereal production by 50 to 70 % by 2100. 

Soil stress‐ Nutrient uptake‐ Contamination‐ Soil‐borne pathogens

Abiotic stress‐ Salinity‐ Drought‐ Heat‐ Frost

Biotic stress‐ Viruses‐Microbes‐ Herbivores‐Weeds

Quality‐ Protein content‐Micronutrients‐ Food Safety‐ Consumer preference

Productivity‐ Grain size‐ Grain number‐ Biomass

Crop improvement and food security

Develop novel genetic resources in crops to improve production of healthy food andto adapt to future environmental conditions. 

Natural diversity Mutagenesis

Beneficial traits are driven by changes in genomes:

Drought susceptible

Drought resistant

Naturally‐occurring modifications in genome

Artificially‐induced randommodifications in genome

X

Beneficial traits and conventional crop breeding:

Drought susceptible

Drought resistant

Phenotypicselection

Ancestral

Breeding

X

Beneficial traits and conventional crop breeding:

Drought susceptible

Drought resistant

Phenotypicselection

Geneticselection

Ancestral

Breeding

Mod

ern

Breeding

The new era: genomics

2001 

2009 

2018 

Genetic region responsible for trait

The new era: genomics

2001 

2009 

2018 

2000 

2002 

Genetic region responsible for trait

…ATGCATGCATGCATGCATGCATGCATGCATGCATGCATGC…Genomic sequence responsible for trait‐ Presence/absence of a gene‐ Subtle modification in a gene sequence

The new tool for crop improvement:genome editing

Genome editing (genome engineering) is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of living organisms.

Unlike early genome engineering techniques (referred as techniques leading to the creation of Genetically Modified Organisms) that randomly insert genetic material into a host genome, genome editing targets DNA changes to specific location in the genome.

There is no stable incorporation of foreign DNA in the genome of the resulting edited plant.The result is similar to mutagenesis (subtle change), but specifically targeted (more efficient, less off‐target impacts) 

Regulatory agencies consider genome edited plants as similar to mutagenized plants, therefore edited plants should not be subjected to GMO regulations.

The CRISPR-Cas9 system and its origin

2015 

Bacterial immune system against virus

Cas genes

CRISPR‐associated (Cas) Clustered Regularly Interspersed Short Palindromic Repeats

Bacterial DNA

The CRISPR-Cas9 system and its origin

2015 

Bacterial immune system against virus

Viral DNA

Cas genes

CRISPR‐associated (Cas) Clustered Regularly Interspersed Short Palindromic Repeats

Bacterial DNA

The CRISPR-Cas9 system and its origin

2015 

Bacterial immune system against virus

Viral DNA

1. memory

Cas genes

CRISPR‐associated (Cas) Clustered Regularly Interspersed Short Palindromic Repeats

Bacterial DNA

The CRISPR-Cas9 system and its origin

2015 

Bacterial immune system against virus

Viral DNA

1. memory2. recognition

Cas genes

CRISPR‐associated (Cas) Clustered Regularly Interspersed Short Palindromic Repeats

Bacterial DNA

The CRISPR-Cas9 system and its origin

2015 

Cas genes

CRISPR‐associated (Cas) Clustered Regularly Interspersed Short Palindromic Repeats

Bacterial immune system against virus

Bacterial DNA

Viral DNA

1. memory3. degradation

2. recognition

The CRISPR-Cas9 system and its origin

2015 

Cas genes

CRISPR‐associated (Cas) Clustered Regularly Interspersed Short Palindromic Repeats

Bacterial immune system against virus

Bacterial DNA

Viral DNA

1. memory

2. recognition

3. degradation

How can CRISPR be used in crop improvement?If we incorporate a CRISPR‐Cas system in plants, and ask plants to target their own DNA,we can specifically induce DNA changes at desired location in the plant genome.

bacterial Cas9

guide RNAs

Plant DNAGene of interest 1

1 2

Gene of interest 2

3 4 5

Examples of ongoing applications in bread wheat

Knocking‐out a gene

Cas 9guide RNAs

1 2 3 4 5 6

ASN2

ASN2

ASN2

Genome A

Genome B

Genome D

Non functional ASN2(low acrylamide content in grain)

‐‐‐deletion‐‐‐Expectedoutcome

Examples of ongoing applications in bread wheat

Knocking‐out a gene

Cas 9guide RNAs

1 2 3 4 5 6

ASN2

ASN2

ASN2

Genome A

Genome B

Genome D

Non functional ASN2(low acrylamide content in grain)

‐‐‐deletion‐‐‐Expectedoutcome

Allele replacement

Cas 9guide RNAs

1 2 resistant ACCase

susceptible ACCaseA/B/D genomes 

A

T

resistant ACCaseA

Subtle change in ACCase to obtain a resistant isoform(tolerance to herbicides based on Fop and Dim chemistries) 

Expectedoutcome

Aresistant ACCase

Replacement DNA

Delivery of the CRISPR components in wheat.

Transgenic approach

Transform wheat plants with a DNA construct which contains all required components (Cas9, guide RNAs, and alternative DNA template when allele replacement).

Transformation protocol for Chinese Spring (model cultivar) available.Transformation protocol for CO winter wheat under development.

Yields to transgenic plants (=GMO) but, transgenic part can be removed from the genome through breeding.

DNAchange

CRISPR‐Cassystem

chr.1 chr.2

Transient transgenic step

selection

DNAchange

chr.1 chr.2

Non‐GMO edited crop

Delivery of the CRISPR components in wheat.

Transgenic approach In vitro approach

Transform wheat plants with a DNA construct which contains all required components (Cas9, guide RNAs, and alternative DNA template when allele replacement).

Transformation protocol for Chinese Spring (model cultivar) available.Transformation protocol for CO winter wheat under development.

Yields to transgenic plants (=GMO) but, transgenic part can be removed from the genome through breeding.

DNAchange

CRISPR‐Cassystem

chr.1 chr.2

Transient transgenic step Wild‐type crop

selection

DNAchange

Incubate wheat plants (embryos) with all required components (Cas9, guide RNAs, and alternative DNA template when allele replacement), all directly synthesized in vitro.

No incorporation of foreign DNA in the plant genome.

Components will be progressively degraded in the plant cell. Does not yield to any GMO intermediate.

DNAchange

chr.1 chr.2

Non‐GMO edited crop Non‐GMO edited crop

A set of target genes for various desired traits

Herbicide tolerance

Nutritional quality

Flour color

ACCase genes

PPO genes

FRO‐HMA genes

Virus resistanceeIF genes

Drought toleranceERA genes

Plant/Grain sizeELA‐DWF  genes

Acrylamide contentASN genes

Acknowledgments

Dr. Ing. Agro. Facundo TabbitaINTA, Argentina.

Andrew Katz(PhD Student)CSU

Elana BoltzUndergrad. StudentCSU

Genome editing

Patricia VailUndergrad. StudentCSU

Jack MentzerUndergrad. StudentCSU

Winter and spring wheat transformation

Funding