Download Wheat Genetics Project: Identification, Characterization, and

Genetic “Engineering” for
Crop Improvement
Xiwen Cai
North Dakota State University, Fargo, ND
Outline
• Definition of genetic engineering
• Genetic engineering at the whole
plant level
• Genetic engineering at the cell level
• Genetic engineering at the
chromosome level
• Genetic engineering at the DNA level
Engineering
Engineering is the application of
scientific, economic, social, and
practical knowledge in order to
invent, design, build, maintain,
research, and improve structures,
machines, devices, systems,
materials, and processes.
wikipedia.org
“Genetic Engineering”
Genetic engineering, also called genetic
modification, is the direct manipulation of an
organism's genome using biotechnology. New
DNA may be inserted in the host genome by
first isolating and copying the genetic material
of interest using molecular cloning methods to
generate a DNA sequence, or by synthesizing
the DNA, and then inserting this construct into
the host organism. An organism that is
generated through genetic engineering is
considered to be a genetically modified
organism (GMO).
wikipedia.org
Genetic Engineering
Genetic engineering, also called genetic
modification, is the manipulation of an
organism's genome at whole plant, cell,
chromosome, and DNA/RNA levels using
conventional and modern genetic,
cytogenetic, and genomic technologies. The
end products of genetic engineering are
genetically modified organisms (GMOs) no
matter how they are modified genetically.
Are “Genetically Modified” Crops
And Foods New?
We have been genetically modifying
plants and animals for a very long
time or since the dawn of civilization!
Almost all crop plants have been
extensively modified genetically
compared to their wild relatives.
Genetic Engineering At
Whole Plant Level
• Conventional breeding – homologous
meiotic recombination-based
- Select parents and make crosses
- Homologous meiotic recombination
- Select recombinants of interest
- Evaluate traits of recombinants
- Release varieties
Genetic Engineering At
Whole Plant Level
• Mutation breeding (perennials and
others with narrow genetic
variation…)
- Induce mutations
- Select mutants of interest
- Evaluate traits of mutants
- Release varieties
Genetic Engineering At Cell
Level
• Cell/tissue culture
- Structural variation in chromosomes and DNA
• Nuclear substitution
- Nucleus-cytoplasm interaction
- Male sterility and heterosis in crop production
Genetic Engineering At
Chromosome Level
• Induce homoeologous meiotic recombination
Goatgrass-Derived Rust Resistance Gene
Genetics 187:1011-1021 (2011)
Genetic Engineering At
Chromosome Level
• Induce chromosome mutations
- Irradiation-induced
Chen et al. 2008. Science in China Series C: Life Sciences:51: 346-352
Genetic Engineering At
Chromosome Level
• Induce chromosome mutations
- Gametocidal chromosome-induced
Downloaded from http://jhered.oxfordjournals.org/ at Huazhong Agricultu
Endo and Gill,
J. Hered 87:295-307
(1996)
Genetic Engineering At
Chromosome Level
• Induce chromosome mutations
- Misdivision-induced
Friebe et al. Cytogenet
Genome Res 109:293–297
(2005)
Genetic Engineering At
Chromosome Level
• Manipulate chromosomal behavior in
meiosis and mitosis
- Meiotic restitution
Genetic Engineering At
Chromosome Level
• Manipulate chromosomal behavior in
meiosis and mitosis
- Wide hybridization-induced chromosome
elimination
e.g. Wheat x Maize hybridization system for
wheat haploid production – doubled
haploid mapping population
Genetic Engineering At
Chromosome Level
• Manipulate ploidy level - haploidy,
diploidy, and polyploidy
- Chemical-induced chromosome doubling
- Meiotic restitution-induced chromosome
doubling
- Wide hybridization-resulted chromosome
elimination
Genetic Engineering At DNA Level
• Transformation
Genetic Engineering At DNA Level
• Viral infection, microinjection, and
bombardment with DNA-coated tungsten
particles
Griffiths et al. 1999
Genetic Engineering At DNA Level
• Genome editing
Artificially engineered nuclease ("molecular scissor”)mediated genome modifications, including insertion,
substitution, deletion, and inversion, etc.
 Create site-specific double-stranded breaks (DSBs) by
engineered nucleases
 Repair DSBs and bring in DNA sequence changes by
homologous recombination (HR) and nonhomologous
end-joining (NHEJ)
Genetic Engineering At DNA Level
• Genome editing
- Create site-specific DSBs
 Engineered nucleases:
 Meganucleases – Recognize long DNA sequence (14-40 bp) and many
engineered versions: less toxicity and high costs
 Zinc finger nucleases (ZFNs) and Transcription Activator-Like Effector
Nucleases (TALENs) – Restriction endonuclease consisting of a specific
DNA-binding domain at the N-terminal and a non-specific DNA cleavage
domain at the C-terminal – Protein-guided
ZFNs are generated by fusing a zinc finger DNA-binding domain to a
DNA-cleavage domain. Zinc finger domains can be engineered to target
specific desired DNA sequences.
TALENs are generated by fusing a TAL effector DNA-binding domain
to a DNA cleavage domain. Transcription activator-like effectors
(TALEs) can be quickly engineered to bind practically any desired
DNA sequence.
wikipedia.org
Genetic Engineering At DNA Level
• Genome editing
- Create site-specific DSBs
 Engineered nucleases:
Protein-guided
nucleases
wikipedia.org
Genetic Engineering At DNA Level
• Genome editing
- Create site-specific DSBs
 Engineered nucleases:
 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas
(CRISPR-associated) system – RNA-guided
Genomic Structures of the CRISPR-Cas Systems in
Streptococcus thermophilus Bacterium
The cas genes are
represented in gray,
and the repeat-spacer
array in black. The
repeat and spacer
(captured phage or
plasmid nucleic acid)
are detailed as black
diamonds (T, terminal
repeat) and white
rectangles,
respectively. Bottom
line, consensus repeat
sequence. L1 to L4,
leader sequences. The
predicted secondary
structure of the
CRISPR3 repeat is
shown on the right.
Science 327: 167-170 (2010)
CRISPR-Cas as an Immune System in Bacteria and Archaea
Overview of the CRISPR/Cas
mechanism of action. (A)
Immunization process: After
insertion of exogenous DNA from
viruses or plasmids, a Cas
complex recognizes foreign DNA
and integrates a novel repeatspacer unit at the leader end of
the CRISPR locus. (B) Immunity
process: The CRISPR repeatspacer array is transcribed into a
pre-crRNA that is processed into
mature crRNAs, which are
subsequently used as a guide by
a Cas complex to interfere with
the corresponding invading
nucleic acid. Repeats are
represented as diamonds,
spacers as rectangles, and the
CRISPR leader is labeled L.
Science 327: 167-170 (2010)
Natural and Engineered CRISPR-Cas9 System
(a) Naturally occurring CRISPR systems
incorporate foreign DNA sequences into
CRISPR arrays, which then produce
crRNAs (CRISPR RNA) bearing
“protospacer” regions that are
complementary to the foreign DNA site.
crRNAs hybridize to tracrRNAs
(transactivating CRISPR RNA; also
encoded by the CRISPR system) and this
pair of RNAs can associate with the Cas9
nuclease. crRNA-tracrRNA:Cas9
complexes recognize and cleave foreign
DNAs bearing the protospacer sequences.
(b) The most widely used engineered
CRISPR-Cas system utilizes a fusion
between a crRNA and part of the tracrRNA
sequence. This single gRNA complexes
with Cas9 to mediate cleavage of target
DNA sites that are complementary to the
5′ 20 nt of the gRNA and that lie next to a
PAM (protospacer adjacent motifs)
sequence. (c) Example sequences of a
crRNA-tracrRNA hybrid and a gRNA.
Nature Biotechnology 32: 347–355 (2014)
Genetic Engineering At DNA Level
• Genome editing
- DSB repair
DSB repair pathways: Non-homologous end joining (NHEJ) and
homology directed repair (HDR)
Nature Biotechnology 32:
347–355 (2014)
Applications of the CRISPR-Cas9 System in Genome
Editing and Other Genome Studies
(a,b) gRNA-directed Cas9
nuclease can induce indel
mutations (a) or specific
sequence replacement or
insertion (b). (c) Pairs of
gRNA-directed Cas9
nucleases can stimulate large
deletions or genomic
rearrangements (e.g.,
inversions or translocations).
(d–f) gRNA-directed dCas9
can be fused to activation
domains (d) to mediate
upregulation of specific
endogenous genes,
heterologous effector domains
(e) to alter histone
modifications or DNA
methylation, or fluorescent
proteins (f) to enable imaging
of specific genomic loci. TSS,
transcription start site.
Nature Biotech 32: 347–355 (2014)
Application of the CRISPR-Cas9 System in Plants
(a) Assay scheme. (b) DNA gel with PCR bands obtained upon amplification using primers flanking the target site
within the PDS gene of N. benthamiana. In lanes 1–3 the template genomic DNA was digested with MlyI, whereas
in lane 4 nondigested genomic DNA was used. (c) Alignment of reads with Cas9-induced indels in PDS obtained
from lane 1 of b. The wild-type sequence is shown at the top. The sequence targeted by the synthetic sgRNA is
shown in red whereas the mutations are shown in blue. PAM, the protospacer-adjacent motif, was selected to
follow the consensus sequence NGG. The changes in length and sequence are shown to the right.
Nature Biotech 31: 691-693 (2013)
Acknowledgements
• Postdoctoral researchers, graduate students,
visiting scientists, technicians, and
undergraduate helpers in my lab
• My collaborators at NDSU, USDA-ARS, and
other institutions in the US and other countries
• Funding agencies:
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USDA-NIFA-AFRI
USWBSI
National Science Foundation
ND Wheat Commission