Transcript CRISPR/CAS9 - Ryerson University

From Plants to Energy:
Efficient
Lignin
Degradation
BIOFUELS
Margarette Francis, Maham Hijazi, Reeda Mahmood,
Riza Unabia & Manaum Zain
1
FOSSIL FUELS: USAGE AND CONCERNS
 “Fossil Fuels” in today’s world
 Green house gasses, climate change and global warming
 Why still use them?
 Economically, it is more beneficial
 Hybrid electric car
 inefficent
2
ALTERNATIVE OPTIONS…
 Biofuels  Ethanol
 Increasingly common option in agricultural and forestry industry in
Canada
 DISADVANTAGE  presence of lignin
 Genomics to overcome this problem
 An exemplary model  SOYBEAN
3
WHY SOYBEAN?
Soybean (Glycine max)
Abundant all over the world
Grown in Southern and Eastern Ontario
Breeding advances (E.O)
Significant source of protein and oil
4
ETHANOL AS AN ENERGY SOURCE…
Reduces greenhouse gases
Does not contribute to carbon dioxide
emissions (34% reduction)
Input of energy required to produce
ethanol has dropped by 36%
Gives more energy compared to other renewable sources
5
WHY IS LIGNIN A BARRIER?
 Barrier for cellulose and hemicellulose extraction
 Lignin removal improve the digestibility of the biomass
 Mechanical and chemical treatments are used to breakdown lignin
 rate of reaction is slow
 Altering the pathway in order to reduce lignin is more effective
 rate of reaction is comparatively faster
Wood Composition. (2007). Retrieved from http://learn.forestbioenergy.net/learning-modules/module-6/unit-1/lesson-1
Yarris, L. (2013, November 13). Lignin-Feasting Microbe Holds Promise for Biofuels. States News Service. Retrieved from http://www.highbeam.com/doc/1G1-348985171.html?
6
THE EFFECT OF LIGNIN REDUCTION ON THE
STRUCTURE OF PLANTS
 Removing too much lignin from a plant affects its phenotype
 Research conducted on the plant Arabidopsis thaliana
 Reduction of 64% of lignin, compensated by the increase in matrix
polysaccharides or cellulose
 Afingen technique was used for lignin reduction,
producing functionally and structurally normal plants
Stems & Vascular Tissue. (2012). Retrieved from http://www.sciencepartners.info/?page_id=534
7
The
Phenylpropanoid
Pathway
Reinprecht, Y., Yadegari, Z., Perry, G., Siddiqua, M., Wright, L., Mcclean, P., & Pauls, K. (2013). In silico comparison of genomic regions containing genes coding for enzymes and transcription factors for the phenylpropanoid pathway in
Phaseolus vulgaris L. and Glycine max L. Merr. Frontiers in Plant Science. Retrieved March 19, 2015, from http://journal.frontiersin.org/article/10.3389/fpls.2013.00317/full
8
A CLOSER LOOK….
Cinnamyl alcohol dehydrogenase (CAD)
catalyzes the reaction that is involved in the
formation of the monolignols that make up
lignin.
CAD
P-Hydoxyphenol lignin
CAD
Guaiacyl lignin
CAD
Syringyl lignin
Baldoni, A., Von Pinho, E. V. R., Fernands, J. S., Abreu, V. M., & Carvalho, M. L. M. (2013). Gene Expression in the lignin biosynthesis pathway during soybean seed development. Genet. Mol. Res 12 (3) 2618-2624
9
METHODS for GENOME EDITING
 Finger nucleases
 RNA interference
 Transcription activator-like effector nucleases
 Expensive and time-consuming
 A recent and more innovative process..
10
CLUSTERED REGULARLY INTERSPACED
SHORT PALINDROMIC REPEATS (CRISPRS)
AND CRISPR-ASSOCIATED (CAS)
PROTEINS
CRISPR/CAS9 SYSTEM
11
WHAT IS IT?
 derived from a natural process found in bacteria to
protect themselves from pathogens
 targets genes for editing and regulating
 comparable to Photoshop
Horizon Licenses Harvard University Gene-Editing Technology. (2013). Drug Discovery & Development
.
12
TO INITIATE GENE MODIFICATION,
sgRNA
(single guide RNA)
Cas9 nuclease
Cas9 complex
13
Protospacer
Adjacent Motif
(PAM)
Target Sequence
Gene of Interest
14
Non-Homologous End
Joining (NHEJ) DNA
repair pathway
Stop Codon
15
SUMMATION: CRISPR/CAS SYSTEM
sgRNA/Cas9
complex binds to
gene
Cas breaks gene
(double strand
breaks)
Induced mutation
in gene sequence
altered gene sequence  dysfunctional CAD gene
16
WHAT MAKES A GOOD BIOFUEL?
1. Greenhouse Gas Reduction Potential
2. Cost-Effectiveness
3. Energy Allocations for Crops
17
ABUNDANCE IN CANADA - ONTARIO
 Soybean is one of
the most widely
used feedstock for
biofuel production.
 Canada’s second
leading crop.
Crop Production Data in Metric Tonnes
Crops
2012
2013
2014
Barley
165,500
134,600
151,300
Corn
8,598,300
9,007,300
7,600,000
Soybean
3,401,900
3,238,600
3,791,100
Canola
61,200
49,900
31,300
Wheat, winter
1,750,000
2,277,900
1,627,500
Statistics Canada. CANSIM table 001-0010
18
ENERGY ALLOCATIONS
WCBA. Canada. http://westerncanadabiodiesel.org/biodiesel/
Soybean has a much higher net energy balance as compared to various
other feedstocks
19
GREENHOUSE GAS EMISSIONS
 According to the Canadian Natural Resources model, GHGenius, soybean
biodiesel and corn ethanol were found to reduce GHG emissions by 50% and 21%,
respectively.
 According to the Greenhouse gases, regulated Emissions and Energy in Transportation
(GREET) model for life cycle analysis, all soybean-derived fuels achieve a significant
reduction (52–107%) in fossil energy use and in petroleum use (more than 85%)
20
WHY IS IT GOOD FOR CANADA?
 Cost-effective
 Eco-friendly
 Sustainable
 Renewable
21