Transcript Document

Methods for Reverse genetics
References:
1. Alonso JM, Ecker JR. Moving forward in reverse: genetic technologies to enable genomewide 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): 29-38
3. Krysan, Young, and Sussman. T-DNA as an insertional mutagen in Arabidopsis. Plant Cell
1999 11(12): 2283-90
4. Li et al. 2001. A fast neutron deletion mutagenesis-based reverse genetics system for
plants. Plant Journal 27(3): 235-42
5. Till et al. 2003. Large-scale discovery of induced point mutations with high-throughput
TILLING. Genome Research 13(3): 524-30
6. Heidi Scholze and Jens Boch, 2011. TAL effectors are remote controls for gene activation.
Current Opinion in Microbiology. Volume 14, Issue 1, February 2011, 47–53
7. 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 CRISPRCas9. Science. 346(6213):1258096-1.
Genetic analysis
It’s all about mutants and their phenotypes!
Forward genetics
• From mutant phenotype to gene, from gene
to protein function
Reverse genetics
• From gene to mutant phenotype, to function
Reverse genetics
What kinds of manipulation do we usually do
to our favorite gene (FG) ?
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
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 promoter
region of Ku70, which encodes a protein involved in DNA repair.
DAYSLEEPER
25 bp Kubox motif
DAYSLEEPER encodes a hAT-like transposase
hAT-like transposon elements
● 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.
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
T-DNA
Q: How would you prove that the
mutant phenotype is caused by
the mutation in DAYSLEEPER?
-co-segregation
-transgene complementation
-obtaining second allele
DAYSLEEPER over-expressers
Slower 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 do we need reverse genetics?
(When to use reverse genetics?)
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 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: cosuppression
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
RNAi: an ancient immune response
against invasion of viruses and other genetic materials
RISC:
RNA-induced silencing complex
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.
Hairpin RNA-induced gene silencing
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.
Silencing of the phytoene desaturase gene
in Arabidopsis by hairpin RNA
PDS
PDS
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.
Virus-induced gene silencing (VIGS)
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.
Silencing of the phytoene desaturase gene
in tobacco by TEV-based VIGS
PDS
Liu, et al. 2002. Plant Journal, 30: 415–429.
transient- vs. stable-integrated gene-silencing
Advantages
Viral-induced
Gene silencing
(VIGS)
Hairpin
transgenes
● Rapid
● easy to use
● applicable to mature plants
● useful for species hard to
generate transgenic plants
● Not restricted by host range
● controllable tissue
specificity
● range of degrees of silencing
Disadvantages
● Host range limitations
● restricted regions of silencing
● viral symptoms superimposed
on silencing phenotype
● Require Transformation
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
T-DNA
Gene A
How do we test whether a plant has a T-DNA in Gene A?
How do you find a T-DNA insertion mutant in a population
of 60,480 transgenic plants?
Pool 9 into one
Pool 25 into one
Pool 9 into one
LB
5’
RB
3’
Arabidopsis 2010 Project
A research program proposed in 2000 to determine
the function of every gene in Arabidopsis by 2010
Somerville C and Somerville, S. 1999. Plant Functional Genomics.
Science 285(5426): 380-383.
Indexed T-DNA knockout lines
- Built using end-rescue and sequencing of individual
T-DNA line in the population.
T-DNA
Gene A
Ends of ~ 300,000 T-DNA lines have been sequenced.
- 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)
Indexed T-DNA knockout lines
Search engine
T-DNA Express
http://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
DeleteageneTM
Fast Neutron Deletion Mutagenesis-based Reverse Genetics Approach for Plants
Fast neutron bombardment
-random deletions
screen libraries of mutants
find target gene deletions
to
OR
5 kb
GA1 Locus
WT
6.4 kb
ga1-3
1.4 kb
Can we detect the presence of ga1-3
in a population of 1,000 plants
by one PCR reaction?
Deletion screen reconstruction
GA1 Locus
Wild type
(6.4 kb)
WT
6.4 kb
ga1-3 band
(1.4 Kb)
ga1-3
1.4 kb
5 kb Deletion
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
Arabidopsis mutant screen
20 mega pools (2592 lines per pool)
WT
Deletion
9 super pools (288 lines per pool)
WT
Deletion
8 pools (36 lines per pool)
WT
Deletion
2 sub pools (18 lines per pool)
WT
Deletion
Individual lines
WT
Deletion
ARABIDOPSIS MYB19
SUPERPOOL
ANALYSIS
MEGAPOOL ANALYSIS
WT
WT
Mutant
POOL ANALYSIS
PLANT ANALYSIS
1.7 kb Deletion
Mutant
WT
WT
Mutant
Mutant
ARABIDOPSIS MYB19
AtMyb19 Deletion Analysis
WT 39371 gcattcttta attcaattg - - - aacaacaaca tgatcatgaa 41090
Mut 39371 gcattctt/
/a tgatcatgaa 41090
WT
3.0 kb
mutant
1.3 kb
DELETION vs. OTHERS
Deletional
Knockout
Insertional
Knockout
RNAi
Knockout
Applicability in
Crop Species
Wide
Limited
Limited
Cost & Time
Inexpensive
& Fast
Slow
& Expensive
Slow
& Expensive
Gene Specificity
Gene-/Tandem
Gene-Specific
GeneSpecific
Family
Tissue Specificity
None
Limited
Possible
Penetrance
High
High
Unreliable
In Arabidopsis, Deleteagene is especially useful for knocking out
small genes and tandem repeats.
TILLING: Targeting Induced Local
Lesions In Genomes
Detection of point mutations in target genes within
mutagenized or natural populations of plants by
heteroduplex analysis
Developed by:
Steve Henikoff., Fred Hutchinson Cancer Inst.
Luca Comai, University. of Washington
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?
CEL I is a single-stranded DNA endonuclease
with a high specificity for mismatches
3’
5’
5’
3’
CEL1 Cleavage
3’
5’
5’
3’
3’
5’
5’
3’
Denature
3’
5’
5’
3’
5’
5’
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’
PCR
3’
5’
5’
3’
5’
3’
heat (denature)
re-nature
3’
5’
3’
5’
5’
3’
5’
3’
3’
5’
3’
5’
5’
3’
CEL1 Cleavage
3’
5’
5’
3’
3’
5’
5’
3’
Denature
3’
5’
5’
3’
5’
5’
Resolve on Li-Cor Gel
LI-COR Scanning Results:
1.0kb
0.8kb
0.2 kb
IR DYE 700
IR DYE 800
A TILLING gel image
www.licor.com
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
TAL effectors are remote controls for gene activation
Heidi Scholze and Jens Boch
Current Opinion in Microbiology
Volume 14, Issue 1, February 2011, Pages 47–53
Dr. Ulla Bonas
Institute of Biology, Dept. of Genetics
Martin- Luther- University
Halle- Wittenberg
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?
DNA
LTPEQVVAIASNIGGKQALETVQRLLPVLCQAHG
A
LTPEQVVAIASNGGGKQALETVQRLLPVLCQAHG
T
LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHG
C
LTPEQVVAIASNKGGKQALETVQRLLPVLCQAHG
G
LTPEQVVAIASNNGGKQALETVQRLLPVLCQAHG
A/G
TAL Nucleases (TALENs): artificial restriction enzymes
Cleave DNA only as dimers
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.
TAL Nucleases (TALENs)-based genome editing
Advantages and disadvantages of using
TALENs for Reverse Genetics
Advantages:
• Targeted editing of a gene of interest
• Potentially applicable to many different species.
Disadvantage:
• Constructs encoding TALENs are complex
and hard to make.
Genome Editing
Using the CRISPR-Cas9 System
CRISPR: clustered regularly interspaced
short palindromic repeats
Cas: CRISPR-associated (cas) genes
Bacterial Immune System
Cas9 (CRISPR associated protein 9)
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.
Can we apply the CRISPR-Cas9 system to
modify target genes in plants?
How?
CRISPR-Cas9: a RNA-guided platform
to cut at specified locations in the genome
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
Guide RNA: crRNA + tracrRNA (trans-activating RNA )
PAM: protospacer adjacent motif, NGG
gRNA target sequence
PAM
AGCTGGGATCAACTATAGCG NGG
9 out of 96 T1 transgenic plants contain mutations
in the rice phytoene desaturase gene
Cleavage site
(1) Wild type rice plant
(2) Monoallelic mutant
(3) Biallelic mutant
(4) Biallelic mutant
BbsI BbsI
LB
RB
pAtU6
Target
seq
sgRNA
scaffold
pAtUBQ
Cas9
tAtUBQ
Hygr
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?