Objective: Convert a hulled (covered) barley into a hull-less (Naked!) barley - PowerPoint Presentation

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Objective: Convert a hulled (covered) barley into a hull-less (Naked!) barley

Taketa PNAS 2008

Hulled phenotype controlled by one gene: NUD

>NUD_CDS

ATGGTACAGTCCAAGAAGAAGTTTCGCGGCGTCAGGCAGCGCCACTGGGGCTCCTGGGTCTCCGAGATCAGGCATCCTCTCCTAAGAGGAGGGTGTGGTTGGGCACCTTTGAGACGGCGGAGGAGGCTGCGCGGGCGTACGATGAGGCTGCCATCCTGATGAGCGGGCGCAACGCCAAGACCAACTTCCCCGTACCGAGGAGTGCCAACGGGGAGATCATCGTCGCCCCAGCAGCAGCAGCACGGGACATTCGCGGTGGCGTTGGCTCGTCGTCCTCCGGGGCCGCCGGCGCCAGCAGCCTGTCACAGATCCTCAGCGCCAAGCTCCGCAAGTGCTGCAAGACACCGTCCCCGTCCCTCACCTGCCTCCGCCTCGACACCGAGAAGTCCCACATTGGCGTCTGGCAGAAGCGCGCGGGTGCCCGTGCCGACTCCAGCTGGGTCATGACCGTCGAGCTCAACAAGGAGCCGGCCGCAGCGGCACCACCAACGCCCAGCGACAGCACGGTGTCGGCGACTCCTTCCTCGTCCACGTCCACGTCCACAACGGGCTCCCCACCGGAGGCAATGGAGGACGAAGAGAGGATCGCGCTGCAGATGATAGAGGAGCTGCTGAGCAGGAGCAGCCCGGCTTCGCCGTCACATGGGCTGCTGCACGGTGAAGAAGGCAGCCTCCTCATCTGA

Disrupting this gene, through inducing deletions or mutations, should cause loss of the hulled phenotype

Cas9 is guided to specific DNA sequences by a so-called “single guide RNA” (

sgRNA)

RNA-guided Cas9-mediated

genome engineering is used to create double stranded breaks

sgRNA

guide target sequence must be followed by a “

Protospacer

Adjacent Motif” (PAM), consisting of NGG

Any 23bp sequence ending ‘GG’ can be targeted by an RNA-guided Cas9

Designing the

sgRNA

5’ 

G

NNNN NNNNN NNNNN NNNNN NGG 3’

PAM sequence (NGG) needs to follow target

Target needs to start with G as the chosen promoter start of transcription is G

Target sequence 20bp

Designing the

sgRNA

5’ 

G

NNNN NNNNN NNNNN NNNNN NGG 3’

For testing

transgenics

, it is useful to have a restriction site spanning the site of the double stranded break

Mutations or deletions following the double stranded break induced by Cas9 will disrupt the restriction site

Step 1: Select targets within NUD

GACGCCGCGAAACTTCTTCTTGG

GCGGCGTCAGGCAGCGCCACTGG

GGCGTCAGGCAGCGCCACTGGGG

GGCAGCGCCACTGGGGCTCCTGG

GCAGCGCCACTGGGGCTCCTGGG

GCTCCTGGGTCTCCGAGATCAGG

…

…

Python script:

identify PAM sequences

take 20bp before

check begins with G

NGG

G

20bp

Target 1

Target 2

Target 3

Target 4

Target 5

Target 6

…

Manual curation:

check targets for restriction sites

Multiple targets can be chosen to create multiple

sgRNAs

Step 1: Chosen targets

>NUD_CDS

ATGGTACAGTCCAAGAAGAAGTTTCGC

GGCGTCAGGCAGCGCCACTGGGGCTCCTGGGTCTCCGAGATCAGGCATCCTCTCCTAAGAGGAGGGTGTGGTTGGGCACCTTTGAGACGGCGGAGGAGGCTGCGC

GGGCGTACGATGAGGCTGCCATCCTGATGAGCGGGCGCAACGCCAAGACCAACTTCCCCGTACCGAGGAGTGCCAACGGGGAGATCATCGTCGCCCCAGCAGCAGCAGCACGGGACATTCGCGGTGGCGTTGGCTCGTCGTCCTCCGGGGCCGCCGGCGCCAGCAGCCTGTCACAGATCCTCAGCGCCAAGCTCCGCAAGTGCTGCAAGACACCGTCCCCGTCCCTCACCTGCCTCCGCCTCGACACCGAGAAGTCCCACATTGGCGTCTGGCAGAAGCGCGCGGGTGCCCGTGCCGACTCCAGCTGGGTCATGACCGTCGAGCTCAACAAGGAGCCGGCCGCAGCGGCACCACCAACGCCCAGCGACAGCACGGTGTCGGCGACTCCTTCCTCGTCCACGTCCACGTCCACAACGGGCTCCCCACCGGAGGCAATGGAGGACGAAGAGAGGATCGCGCTGCAGATGATAGAGGAGCTGCTGAGCAGGAGCAGCCCGGCTTCGCCGTCACATGGGCTGCTGCACGGTGAAGAAGGCAGCCTCCTCATCTGA

PAM

PAM

target

target

Target 1

Target 2

Restriction sites

Expected deletion in bold

New England

BioLabs

https://

www.neb.com

/products/e1600-neb-golden-gate-assembly-mix#Product%20Information

Overview of Golden Gate assembly

Golden Gate Assembly allows for the efficient assembly of DNA fragments using sequential or simultaneous digestion and ligation reactions.

A Type IIS restriction enzyme, such as

BsaI

, creates DNA fragments with unique overhangs, and a T4 DNA ligase is used to assemble the fragments together.

Step 2: Synthesize chosen primers for Golden Gate assembly

tgt

ggtctc

aA

TTGNNNNNNNNNNNNNNNNNNN

gttttagagctagaaatagcaag

tgt

ggtctc

a

AGCG

TAATGCCAACTTTGTAC

Forward

Reverse

Target sequence 20bp

Digestion site

(

BsaI

)

Additional flanking sequence to target is for use as primer for amplification from plasmid

note: PAM site is not part of the target sequence

Complementary sequence to anneal to

sgRNA

plasmid

Step 3: Amplify a unique

sgRNA

using primer pair

Amplify from plasmid containing the

sgRNA

scaffold

PCR product:

tgt

ggtctc

a

ATTG

NNNNNNNNNNNNNNNNNNN

GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTACAAAGTTGGCATTA

CGCT

t

gagacc

aca

Target sequence 20bp

BsaI

site

BsaI

site (reverse complement)

Overhang sequences used for Golden Gate Assembly

ALTERNATIVELY (combine step 2 and 3)

PCR product:

tgt

ggtctc

a

ATTG

NNNNNNNNNNNNNNNNNNN

GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTACAAAGTTGGCATTA

CGCT

t

gagacc

aca

Target sequence 20bp

BsaI

site

BsaI

site (reverse complement)

Synthesize

the whole

sgRNA

including desired target sequence

Overhang sequences used for Golden Gate Assembly

Step 4: Assemble level 1

sgRNA

expression cassette using Golden Gate Assembly

Set up digestion-ligation reaction with:

PCR amplicon

Plasmid containing Level 0 wheat U6 promoter

Level 1 acceptor plasmid

Ta

NB. This step can be repeated to create multiple

sgRNAs

Level 0

Initial plasmid containing U6 promoter from wheat

Step 4: Assemble level 1

sgRNA

expression cassette using Golden Gate Assembly

Ta

Level 1 Acceptor plasmid

pICH47732

Description: Position 1

Comments: 

AddGene #48000Inside overhang L: 

GGAG

Inside overhang R: 

CGCT

Resistance: Amp

LacZ

can be used as selectable marker as should be swapped for insert

Step 4: Assemble level 1

sgRNA

expression cassette using Golden Gate Assembly

Recap

PCR amplicon

Plasmid containing Level 0 wheat U6 promoter

Level 1 acceptor plasmid

Forward primer

Reverse primer

plasmid containing the

sgRNA

scaffold

Ta

Step 5: Assemble Level 2

Selectable marker

Cas9 cassette

Multiple

sgRNAs

can be added

Step 6: Transform

Agrobacterium

with final level 2 construct

Assembled

sgRNAs

are combined with a selectable marker and a Cas9 cassette into a Level 2 Acceptor.

This creates an expression cassette ready for transformation into

Agrobacterium

, and subsequent barley transformation.

Step 7: Transformation of barley

Immature embryos are harvested

from immature seed and their embryonic axis is removed

The transformed

Agrobacterium

is dropped on to the embryo

Harwood

et al

. (2009) Barley Transformation Using Agrobacterium-Mediated Techniques

Transformation of barley

Embryos transferred to callus induction plates

Plated on regeneration media containing selection

Transgenic barley transferred to culture tube

Plants left to develop before potting into soil

Harwood

et al

. (2009) Barley Transformation Using Agrobacterium-Mediated Techniques

Step 8: Confirmation of transgenic barley

Carry out DNA extraction of

transgenics

PCR amplify the site of

sgRNA

target using flanking markers

Forward primer

Reverse primer

sgRNA

1

sgRNA

2

>NUD_CDS

ATGGTACAGTCCAAGAAGAAGTTTCGC

GGCGTCAGGCAGCGCCACTGGGGCTCCTGGGTCTCCGAGATCAGGCATCCTCTCCTAAGAGGAGGGTGTGGTTGGGCACCTTTGAGACGGCGGAGGAGGCTGCGC

GGGCGTACGATGAGGCTGCCATCCTGATGAGCGGGCGCAACGCCAAGACCAACTTCCCCGTACCGAGGAGTGCCAACGGGGAGATCATCGTCGCCCCAGCAGCAGCAGCACGGGACATTCGCGGTGGCGTTGGCTCGTCGTCCTCCGGGGCCGCCGGCGCCAGCAGCCTGTCACAGATCCTCAGCGCCAAGCTCCGCAAGTGCTGCAAGACACCGTCCCCGTCCCTCACCTGCCTCCGCCTCGACACCGAGAAGTCCCACATTGGCGTCTGGCAGAAGCGCGCGGGTGCCCGTGCCGACTCCAGCTGGGTCATGACCGTCGAGCTCAACAAGGAGCCGGCCGCAGCGGCACCACCAACGCCCAGCGACAGCACGGTGTCGGCGACTCCTTCCTCGTCCACGTCCACGTCCACAACGGGCTCCCCACCGGAGGCAATGGAGGACGAAGAGAGGATCGCGCTGCAGATGATAGAGGAGCTGCTGAGCAGGAGCAGCCCGGCTTCGCCGTCACATGGGCTGCTGCACGGTGAAGAAGGCAGCCTCCTCATCTGA

Run PCR products on gel against wild type control

WT

1

2

If see PCR product from transgenic samples is smaller than wild type, then deletion of sequence has occurred.

WT

1

2

If bands similar size, deletion or mutation is not easy to detect.

Then need to digest the PCR product.

Digest PCR sample using restriction enzymes

PAM

PAM

target

target

Restriction sites

>

NUD_target_region

ATGGTACAGTCCAAGAAGAAGTTTCGC

GGCGTCAGGCAGC

GCCACTGGGGC

TCCTGGGTCTCCGAGATCAGGCATCCTCTCCTAAGAGGAGGGTGTGGTTGGGCACCTTTGAGACGGCGGAGGAGGCTGCGC

GGGCGTACGATGAGGCTGCCATCCTGATGAGCGGGCGCAACGCCAAGACCAACTTCCCCGTACCGAGGAGTGCCA

BgII

WT

1

2

Digestion with

BgII

PCR product will not be digested if there is disruption of the restriction site.

Therefore, the gene has been disrupted.

Digest PCR sample using restriction enzymes

>

NUD_target_region

ATGGTACAGTCCAAGAAGAAGTTTCGC

GGCGTCAGGCAGC

GCCACTG

GGGC

TCCTGGGTCTCCGAGATCAGGCATCCTCTCCTAAGAGGAGGGTGTGGTTGGGCACCTTTGAGACGGCGGAGGAGGCTGCGC

GGGCGTACGATGAGGCTGCCATCCTGATGAGCGGGCGCAACGCCAAGACCAACTTCCCCGTACCGAGGAGTGCCA

BgII

Confirmed

transgenics

can be used for experimentation