References:1. Alonso JM, Ecker JR. Moving forward in reverse: genetic technologies to enable genome- wide phenomic screens in Arabidopsis. Nat Rev Genet. 2006 Jul;7(7):524-36.2. Waterhouse and Helliwell. Exploring plant genomes by RNA-induced gene silencing. Nat

Rev Genet. 2003 4(1): 29-383. Krysan, Young, and Sussman. T-DNA as an insertional mutagen in Arabidopsis. Plant Cell

1999 11(12): 2283-904. Li et al. 2001. A fast neutron deletion mutagenesis-based reverse genetics system for

plants. Plant Journal 27(3): 235-425. Till et al. 2003. Large-scale discovery of induced point mutations with high-throughput

TILLING. Genome Research 13(3): 524-306. Heidi Scholze and Jens Boch, 2011. TAL effectors are remote controls for gene activation.

Current Opinion in Microbiology. Volume 14, Issue 1, February 2011, 47–537. Shan et al. 2013. Targeted genome modification of crop plants using a CRISPR-Cas

system. Nat Biotechnol. 31(8):686-8. 8. Doudna and Charpentier E. 2014. The new frontier of genome engineering with CRISPR-

Cas9. Science. 346(6213):1258096-1.

Methods for Reverse genetics

Genetic analysisIt’s all about mutants and their phenotypes!

Forward genetics

• From mutant phenotype to gene, from geneto protein function

Reverse genetics

• From gene to mutant phenotype, to function

Reverse genetics

Knockout analysis:

1. Find/Generate a knockout mutant in FG

2. Analyze the mutant to see if there is any defects

3. Connect the defects with biological processes

What kinds of manipulation do we usually do to our favorite gene (FG) ?

Reverse genetics

Overexpression analysis/ectopic expression

1. Overexpress FG (endogenous promoter)Ectopic expression (CaMV 35S promoter)

2. Analyze the overexpresser to see if there are any defects/phenotypes

3. Connect the defects with biological processes

Over-expression of a gene of interest does not necessarily lead to a gain-of-function effect.

Why?

Story on DAYSLEEPER

Background: DAYSLEEPER was found to bind to the promoterregion of Ku70, which encodes a protein involved in DNA repair.

DAYSLEEPER

25 bp Kubox motif

DAYSLEEPER encodes a hAT-like transposase

● In Arabidopsis, there are 246 hAT-like elements.

● Active hAT transposons: 8bp duplication of the insertion site and short terminal inverted repeats (TIR).

● Fossil elements lack the duplication of the insertion site and TIR, and often are transcriptionally silent.

● Daysleeper is a fossil element, but is expressed.

hAT-like transposon elements

Q: DNA repair -----????----- Transposon

How do you find out the function of DAYSLEEPER?

Q: What would a mutant of a fossil transposable element look like?

DAYSLEEPER knockout mutant

Q: How would you prove that the mutant phenotype is caused by the mutation in DAYSLEEPER?

-co-segregation-transgene complementation-obtaining second allele

T-DNA

DAYSLEEPER over-expressersSlower growth, delayed flowering, altered leaves, etc.

DAYSLEEPER is essential for plant development.

Fossil elements are not always fossils, they have the potential to evolve functions essential for plant growth and development.

Bundock P, Hooykaas P. An Arabidopsis hAT-like transposase is essential for plant development. Nature. 2005 Jul 14;436(7048):282-4.

Why cannot forward genetics find mutants in all the genes?

Somerville C and Somerville, S. 1999. Plant Functional Genomics. Science 285(5426): 380-383.

Why do we need reverse genetics?(When to use reverse genetics?)

Why cannot forward genetics find mutants carrying mutations in all genes?

1. Redundancy

2. Lethal mutations

3. Subtle or not obvious phenotypes

4. Mutant missed from forward genetic screens

Why do we need reverse genetics?(When to use reverse genetics?)

1. Figure out function of YFG

2. Redundant genes

3. Essential genes

4. Assist forward genetics: second allele

Why do we still do forward genetics?

- Process more specific, it is less predictable in reverse genetics

- No previous knowledge needed for forward genetics

- Suppressor or enhancer screens that may lead to new biology

With complete genome sequence information, we can pick and study our favorite genes by reverse genetics.

How do we knockout genes in plants?

Homologous recombination

Not for plants!

Reverse genetics: Gene Knockout Strategies

1. RNAi-based silencing

2. T-DNA or transposon based insertional

mutagenesis

3. Deleteagene

4. TILLING

5. TAL effector-mediated DNA modifications

6. CRISPR-CAS9-based gene editing

RNAi based methods

History:

Early 1990’s, phenomena first found by plant scientists: co-suppression

1998, in C.elegans, formally discover dsRNA as signal for RNA interference (Fire and Mello)

1999, small RNA species derived from mRNA detected (Baulcomb)

2001, discovery of dsRNA processing enzyme Dicer

2006, A. Fire and C. Mello won Nobel prize in medicine because of their discovery of dsRNA as mediator of RNAi

dsRNA-directed gene silencing mechanisms. Short dsRNA molecules can either be expressed by endogenous genes, invading viruses or by experimental means and are funnelled into one of two different silencing mechanisms. siRNAs that are perfectly complementary to their cognate mRNA species induce their endonucleolytic cleavage and degradation. Amplification of the RNAi signal by RDRP-dependent mechanisms, RNA-induced epigenetic control of gene expression as well as RNAi transfer between cells have been observed in some but not all species.

RNAi: an ancient immune response against invasion of viruses and other genetic materials

RISC:RNA-induced silencing complex

A typical T-DNA plasmid for the expression of hairpin RNAs (hpRNAs). A generic silencing precursor construct (pHANNIBAL) that enables hpRNA vectors to be easily constructed has different multiple cloning sites either side of the intron to enable the rapid insertion of target sequences in forward and reverse orientations. 35S, CaMV 35S promoter; Term, transcription termination sequence.

Hairpin RNA-induced gene silencing

                                                                                                                                                                            

Figure 5 | Degrees of silencing produced by hairpin-RNA-encoding transgenes.   The stable transformation of Arabidopsis plants with the same hairpin RNA (hpRNA) construct that is targeted against phytoene desaturase gives rise to lines that show a heritable photobleaching phenotype in: a | all tissues; b | sectors of tissue; or c | the cotyledons, but not the rest of the plant. Images courtesy of C.A.H. and P.M.W., CSIRO, Australia. Reproduced with permission from Ref. 63 © (2002) CSIRO Publishing.

Silencing of the phytoene desaturase gene in Arabidopsis by hairpin RNA

PDS PDS

The tobacco rattle virus (TRV) virus-induced gene-silencing (VIGS) system. Two T-DNA plasmids that encode the TRV genome (one encoding TRV RNA1 and the other encoding TRV RNA2, which carries the inserted target sequence) are propagated separately in Agrobacterium and used to co-infect plant tissue. 35S, CaMV 35S promoter; CP, coat protein; M1,2,3, movement proteins 1, 2, 3; RdRP, RNA-dependent RNA polymerase; Term, transcription termination sequence.

Virus-induced gene silencing (VIGS)

Silencing of the phytoene desaturase gene in tobacco by TEV-based VIGS

Liu, et al. 2002. Plant Journal, 30: 415–429.

PDS

transient- vs. stable-integrated gene-silencing

Viral-induced Gene silencing(VIGS)

● Rapid ● easy to use ● applicable to mature plants ● useful for species hard to

generate transgenic plants

● Host range limitations ● restricted regions of silencing ● viral symptoms superimposed on silencing phenotype

Hairpin transgenes

● Not restricted by host range ● controllable tissue specificity ● range of degrees of silencing

● Require Transformation

Advantages Disadvantages

Reverse genetics: Gene Knockout Strategies

1. RNAi-based

2. T-DNA or transposon based insertional

mutagenesis

3. Deleteagene

4. TILLING

5. TAL effector-mediated DNA modifications

(TALENS)

6. CRISPR-CAS9-based gene editing

Agrobacterium Ti plasmid-based transformation

How do you find a T-DNA insertion mutant in a population of 60,480 transgenic plants?

Gene A

T-DNA

How do we test whether a plant has a T-DNA in Gene A?

Pool 9 into one

Pool 25 into one

Pool 9 into one

LB RB

5’ 3’

Somerville C and Somerville, S. 1999. Plant Functional Genomics. Science 285(5426): 380-383.

Arabidopsis 2010 Project

A research program proposed in 2000 to determine the function of every gene in Arabidopsis by 2010

Indexed T-DNA knockout lines

- Major sources: SALK Institute (SALK lines, USA) Syngenta Inc. (SAIL lines, USA) Wisc lines (UW, Madison lines, USA)

FLAG lines (French)GABI lines (German)SK lines (Canadian)

- Built using end-rescue and sequencing of individual T-DNA line in the population.

Gene A

T-DNA

Ends of ~ 300,000 T-DNA lines have been sequenced.

Indexed T-DNA knockout linesSearch engine

T-DNA Expresshttp://signal.salk.edu/cgi-bin/tdnaexpress

Forward genetics in a reverse way

Alonso JM, Ecker JR. Moving forward in reverse: genetic technologies to enable genome-wide phenomic screens in Arabidopsis. Nat Rev Genet. 2006 Jul;7(7):524-36.

In Arabidopsis, about 300,000 T-DNA lines have been sequenced. T-DNA insertions are still not found in some genes.

Why?

Bigger genes have better chance being knocked out by T-DNA.

Reverse genetics: Gene Knockout Strategies

1. RNAi-based

2. T-DNA or transposon based insertional

mutagenesis

3. Deleteagene: useful for small genes and

tandem repeats

Fast neutron bombardment-random deletions

5 kb

ORscreen libraries of mutants to find target gene deletions

DeleteageneTM

Fast Neutron Deletion Mutagenesis-based Reverse Genetics Approach for Plants

GA1 Locus

1.4 kb

6.4 kb

WT

ga1-3

Can we detect the presence of ga1-3 in a population of 1,000 plants

by one PCR reaction?

Deletion screen reconstruction

1:1

0000

ga1-3 band (1.4 Kb)

5 kb Deletion

1:1

0

1:1

000

1:1

00

Wild type (6.4 kb)

GA1 Locus

1.4 kb

6.4 kb

WT

ga1-3

Deletion library construction

Treat wild type seeds with fast neutron

Plant M1 seeds and grow up population

Collect M2 seeds from individual plants

Plant some seeds from each line

Collect tissue and extract DNA

DeleteageneTM

Fast Neutron Deletion Mutagenesis-based Reverse Genetics Approach for Plants

WTDeletion

WTDeletion

WTDeletion

WTDeletion

WTDeletion

20 mega pools (2592 lines per pool)

9 super pools (288 lines per pool)

8 pools (36 lines per pool)

2 sub pools (18 lines per pool)

Individual lines

Arabidopsis mutant screen

MEGAPOOL ANALYSIS

WT

Mutant

ARABIDOPSIS MYB19SUPERPOOL ANALYSIS

WT

Mutant

WT

Mutant

POOL ANALYSISPLANT ANALYSIS

WT

Mutant

1.7 kb Deletion

1.3 kb

3.0 kbWT

mutant

WT 39371 gcattcttta attcaattg - - - aacaacaaca tgatcatgaa 41090Mut 39371 gcattctt/ /a tgatcatgaa 41090

AtMyb19 Deletion Analysis

ARABIDOPSIS MYB19

Deletional

Knockout

Insertional

Knockout

RNAi

Knockout

Applicability in

Crop SpeciesWide Limited Limited

Cost & TimeInexpensive

& Fast

Slow

& Expensive

Slow

& Expensive

Gene SpecificityGene-/Tandem

Gene-Specific

Gene-

SpecificFamily

Tissue Specificity None Limited Possible

Penetrance High High Unreliable

DELETION vs. OTHERS

In Arabidopsis, Deleteagene is especially useful for knocking out small genes and tandem repeats.

TILLING: Targeting Induced Local Lesions In Genomes

Developed by:

Steve Henikoff., Fred Hutchinson Cancer Inst.

Luca Comai, University. of Washington

Detection of point mutations in target genes within

mutagenized or natural populations of plants by

heteroduplex analysis

Arabidopsis EMS Mutagenesis

• Mutation frequency can be as high as • 500 mutations/genome or

• 1 mutation/1000 bp/ 300 plants

• 5% truncations, 50% missense, 45% silent

How do we determine whether there is a mutation in our gene of interest in a plant?

How do we determine whether there is a mutation in our gene of interest in 3,000

plants?

CEL1 Cleavage

Denature

5’ 3’

3’ 5’

5’ 3’

3’ 5’

3’

3’ 5’5’

5’ 3’

5’

3’ 5’

5’

CEL I is a single-stranded DNA endonucleasewith a high specificity for mismatches

Arabidopsis EMS Mutagenesis

Greene, et al., 2003, Genetics 164: 731-740

PCR Amplification of Target Gene from Pooled Genomic DNA

3’5’

5’3’

5’3’

3’5’

5’ 3’3’ 5’

5’ 3’3’ 5’

5’ 3’

3’ 5’5’ 3’

3’ 5’

PCR

heat (denature) re-nature

CEL1 Cleavage

Denature

Resolve on Li-Cor Gel

5’ 3’

3’ 5’

5’ 3’

3’ 5’

3’

3’ 5’5’

5’ 3’

5’

3’ 5’

5’

Norm

al

Mut

ant

LI-COR Scanning Results:

Norm

al

Mut

ant

IR DYE 700 IR DYE 800

1.0kb

0.8kb

0.2 kb

www.licor.com

A TILLING gel image

Colbert et al.2001, Plant Physiol. 126:480-84

Advantages of TILLING as an Approach for Reverse Genetics

• Mutagenized plants have a large number of randomly distributed mutations per plant genome.

• No transgenic manipulations required.

• Both nonsense (knockout) and mis-sense mutations can be recovered.

• Plants heterozygous for a mutation can be detected (lethality not a problem).

Question

If you are given $5,000,000 of funding today to provide mutants with point mutations to the Arabidopsis research community, how would you do it?

Reverse genetics: Gene Knockout Strategies

1. RNAi-based

2. T-DNA or transposon based insertional

mutagenesis

3. Deleteagene

4. TILLING

5. TAL effector-mediated DNA modifications

(TALENS)

6. CRISPR-CAS9-based gene editing

Dr. Ulla BonasInstitute of Biology, Dept. of GeneticsMartin- Luther- UniversityHalle- Wittenberg

TAL effectors are remote controls for gene activationHeidi Scholze and Jens Boch Current Opinion in MicrobiologyVolume 14, Issue 1, February 2011, Pages 47–53

Xanthomonas campestris pv. vesicatoria (Xcv)Bacterial spot of tomato and pepper

Pepper Resistance gene BS3 confers strong resistance against Xcv

Image: http://www.monsanto.com

Xcv Type III secretion system (T3SS)secretes effectors to disturb host immunity

The transcription factor activity of AvrBs3 elicits different responses in resistant and susceptible plants.AvrBs3 is delivered into host cells via the Xanthomonas type III secretion system (T3SS). In susceptible pepper plants (left, green background), AvrBs3 binds the upa box and activates transcription of upa20, which encodes a basic helix–loop–helix transcription factor. Upa20 then activates transcription of genes like upa7, which together give rise to cellular hypertrophy. In resistant pepper plants (right, yellow background) AvrBs3 binds the Bs3 upa box and activates Bs3 transcription. Bs3 initiates a cell death response either through recognition by a guard protein or by modification of an unidentified interacting protein.

UPA20: cell size regulator; Hypertrophy: increase in cell size

AvrBs3: a Transcription activator-like effector (TALE)

Can we apply what we have learned from AvrBS3 to design DNA-binding proteins for a target DNA

sequence?

LTPEQVVAIASNIGGKQALETVQRLLPVLCQAHG

LTPEQVVAIASNGGGKQALETVQRLLPVLCQAHG

LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHG

LTPEQVVAIASNNGGKQALETVQRLLPVLCQAHG

LTPEQVVAIASNKGGKQALETVQRLLPVLCQAHG

DNA

A

T

C

G

A/G

TAL Nucleases (TALENs): artificial restriction enzymes

TAL nucleases (TALNs) promote genome editing. (a) TALNs are fusions between TAL effectors and the FokI endonuclease domain. A tailored TAL repeat domain controls DNA-binding specificity. (b) Two TALNs bind neighboring DNA boxes and FokI dimerization induces DNA cleavage in the spacer region between the boxes. DNA double-strand breaks can promote nonhomologous end-joining (NHEJ) or homologous DNA recombination (HDR) enabling targeted genome modifications like deletions or insertions.

Cleave DNA only as dimers

TAL Nucleases (TALENs)-based genome editing

Advantages and disadvantages of using TALENs for Reverse Genetics

• Targeted editing of a gene of interest

• Potentially applicable to many different species.

Advantages:

• Constructs encoding TALENs are complex

and hard to make.

Disadvantage:

Genome Editing Using the CRISPR-Cas9 System

CRISPR: clustered regularly interspaced short palindromic repeats

Cas: CRISPR-associated (cas) genes

Bacterial Immune System

Can we apply the CRISPR-Cas9 system to modify target genes in plants?

How?

Cas9 encodes a DNA endonuclease that associates with crRNA (also called guide RNA).

Cas9 unwinds foreign DNA to check if it is complementary to the 20 base pair spacer region of the guide RNA.

Cas9 cleaves the invading DNA if the DNA substrate is complementary to guide RNA.

Cas9 (CRISPR associated protein 9)

1. Design Short guide RNAs with homology to target loci

2. Guide RNA + Cas9 are expressed in the cell

3. The Cas9 cleavage site is repaired by either NHEJ or HDR in tandem with a donor

4. High efficiencies of knockout or knock-in

CRISPR-Cas9: a RNA-guided platform

to cut at specified locations in the genome

Guide RNA: crRNA + tracrRNA (trans-activating RNA )

PAM: protospacer adjacent motif, NGG

AGCTGGGATCAACTATAGCG NGGgRNA target sequence PAM

Cleavage site

9 out of 96 T1 transgenic plants contain mutations in the rice phytoene desaturase gene

(1) Wild type rice plant(2) Monoallelic mutant(3) Biallelic mutant (4) Biallelic mutant

LB

HygrHygrpAtUBQpAtUBQ tAtUBQtAtUBQCas9Cas9sgRNA

scaffoldsgRNAscaffold

BbsIBbsI

pAtU6pAtU6 Target seq

Target seq

RB

What are the advantages of the CRISPR-Cas9 system?

1. Targeted editing of a gene of interest

2. Applicable to crop plants

3. Easy to carry out

4. Target multiple homologous genes

A graduate student found two Arabidopsis genes (PTN1 and PTN2), which encode two related proteinases with 70% sequence identity at amino acid level. She wants to test whether these they are involved in chloroplast development. What would you suggest her to do?

There is no mutant phenotype in the knockout mutants of ptn1 or ptn2. The two genes are next to each other on the chromosome. What would you suggest her to do next?

Using Deleteagene, a mutant with both genes deleted was identified, but only heterozygous mutant plants can be identified. What would you suggest her to do next?