CG920 Genomics
Lesson 12
Practical Applications
Jan Hejátko
Functional Genomics and Proteomics of Plants,
CEITEC - Central European Institute of Technology
And
National Centre for Bimolecular Research,
Faculty of Science,
Masaryk University, Brno
hejatko@sci.muni.cz, www.ceitec.eu
2
Literature
Broughton, J.P., Deng, X., Yu, G., Fasching, C.L., Servellita, V., Singh, J., Miao, X., Streithorst,
J.A., Granados, A., Sotomayor‐Gonzalez, A., Zorn, K., Gopez, A., Hsu, E., Gu, W., Miller,
S., Pan, C.Y., Guevara, H., Wadford, D.A., Chen, J.S., and Chiu, C.Y. (2020). CRISPR‐Cas12‐
based detection of SARS‐CoV‐2. Nat Biotechnol 38, 870‐874.
Dietel, M., and Sers, C. (2006). Personalized medicine and development of targeted therapies:
The upcoming challenge for diagnostic molecular pathology. A review. Virchows Arch 448,
744‐755.
Gaudelli, N.M., Komor, A.C., Rees, H.A., Packer, M.S., Badran, A.H., Bryson, D.I., and Liu, D.R.
(2017). Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage.
Nature 551, 464‐471.
Goh, K.I., Cusick, M.E., Valle, D., Childs, B., Vidal, M., and Barabasi, A.L. (2007). The human
disease network. Proc Natl Acad Sci U S A 104, 8685‐8690.
Chen, J.S., Ma, E., Harrington, L.B., Da Costa, M., Tian, X., Palefsky, J.M., and Doudna, J.A.
(2018). CRISPR‐Cas12a target binding unleashes indiscriminate single‐stranded DNase
activity. Science 360, 436‐439.
Koblan, L.W., Erdos, M.R., Wilson, C., Cabral, W.A., Levy, J.M., Xiong, Z.M., Tavarez, U.L.,
Davison, L.M., Gete, Y.G., Mao, X., Newby, G.A., Doherty, S.P., Narisu, N., Sheng, Q.,
Krilow, C., Lin, C.Y., Gordon, L.B., Cao, K., Collins, F.S., Brown, J.D., and Liu, D.R. (2021). In
vivo base editing rescues Hutchinson‐Gilford progeria syndrome in mice. Nature.
Li, X., Qian, X., Wang, B., Xia, Y., Zheng, Y., Du, L., Xu, D., Xing, D., DePinho, R.A., and Lu, Z.
(2020). Programmable base editing of mutated TERT promoter inhibits brain tumour
growth. Nat Cell Biol 22, 282‐288.
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 Medicine
 Molecular Diagnosis
 Personalized Medicine
 Gene Therapy
 Biotechnology
 Genetically Modified Organisms
 Transgenosis
 Genome Editing
 Model Organisms
 Principles of PCR
Outline
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 Medicine
 Molecular Diagnosis
Outline
5
Molecular Diagnosis
 around 10,000 disorders in humans resulting from a single
mutation
 cystic fibrosis
 sickle cell disease
 muscular dystrophy
 beta thalassemia
 ….
 Early molecular diagnosis
 mutations or infections
 PCR
 DNA (chip) hybridization
 Cas-based
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Molecular Diagnosis
 Mammoth Biosciences
 Co-founded by Jenifer Doudna https://youtu.be/IPe4IdgKGdQ
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 Medicine
 Molecular Diagnosis
 Personalized Medicine
Outline
8
Personalized Medicine
 uses knowledge of the genome for:
 prediction of health risks
 diagnosis
 selection of the most appropriate type of
treatment
 minimizing the side effects of treatment
 prevention
http://www.personalizedmedicinecoalition.org/
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http://www.personalizedmedicinecoalition.org/
Personalized Medicine
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Personalized Medicine
11
http://www.personalizedmedicinecoalition.org/
Personalized Medicine
12
 Problem:
 multigene conditionality of most human diseases
Goh et al., 2007
Personalized Medicine
13
 Problem solving
 systems biology - uses e.g. gene clustering to identify genes involved in
the observed phenomenon
Topotecan-
resistant
Topotecansensitive
Dietel and Sers, 2006
Personalized Medicine
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 Problem solving
 biomarkers
 tests
The Case for Personalized Medicine, 3rd edition
Personalized Medicine
15
 Other problems
 Ethical Issues
 the condition is genetic testing or knowledge of the genome easily
abused
 risk: insufficient data security
 in some countries, employers or insurance companies do not have
access to such data
 High Costs
 medicine could be divided into first-class and low-class services
 globalization gap could grow even larger - poor countries would not
be able to afford this
 Privacy
 crucial and complex issue
 what information about oneself can/should be considered private?
Personalized Medicine
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 Medicine
 Molecular Diagnosis
 Personalized Medicine
 Gene Therapy
Outline
17
Procedure in which the DNA sequence is inserted into
the patient genome to replace or supplement the original
gene
 Options:
 replace the mutated gene
 repair the mutation
 deliver DNA encoding a therapeutic protein
 antisense therapy
 In the future useful for treating e.g. hereditary diseases
 Types:
 somatic gene therapy
 gene therapy of germ cells
Gene Therapy
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 Methods
 viral vectors
 retroviruses
 adenoviruses
 herpes simplex virus
 non-viral methods
 injection of plasmid DNA into muscle
 increased efficiency of DNA delivery
 electroporation
 sonoporation
 „gene gun“ (biolistic)
 magnetofection
 genome editing
Gene Therapy
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Procedure in which the DNA sequence is inserted into
the patient genome to replace or supplement the original
gene
 Options:
 replace the mutated gene
 repair the mutation
 deliver DNA encoding a therapeutic protein
 antisense therapy
 In the future useful for treating e.g. hereditary diseases
 Types:
 somatic gene therapy
 gene therapy of germ cells
Gene Therapy
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Gene Therapy
 Hutchinson–Gilford progeria
syndrome
 C•G-to-T•A mutace (c.1824 C>T;
p.G608G) v genu pro laminin (LMNA)
 Defekt v sestřihu RNA vede k tvorbě
toxického proteoinu progerinu
 Věk dožití cca 14 let
 In vivo oprava pomocí ABEs potvrzena
u myší a lidských fibroblastů (Koblan et
al., 2021)
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Adenine Base Editors
Gaudeli et al., 2017
22
Adenine Base Editors
Kuraoka, 2005
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Ethical Issues
 International Commission on the Clinical Use of Human
Germline Genome Editing
 convened by the U.S. National Academy of Medicine (NAM), the U.S.
National Academy of Sciences (NAS), and the Royal Society of the U.K. …
 …to identify a number of scientific, medical, and ethical requirements that
should be considered, and could inform the development of a potential
pathway from research to clinical use — if society concludes that heritable
human genome editing applications are acceptable
 more details at https://nationalacademies.org/gene-editing/international-
commission/index.htm
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Ethical Issues
 Alliance for Regenerative Medicine
 international group representing the cell and gene therapy sector
 put out a “statement of principles” on genome editing endorsed by 13 of the
most active companies in this field
 changing heritable DNA in sperm, eggs or a new embryo — came true in
November 2018 when He Jiankui, a Chinese biophysicist, said that his lab had
edited two baby girls to make them resistant to HIV infection. This mutation will
be inherited by their descendants.
 31 clinical trials for gene edited therapies are in progress around the world, 20
of which are in oncology. None is yet close to commercialisation. The US has
the largest number of trials (19) followed by China (10) and the UK (6)
FT, Clive Cookson, Science Editor August 27 2019
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Ethical Issues
 Genome editing as a bioweapon?
 ongoing research program funded by the U.S. Defense Advanced Research
Projects Agency (DARPA)
 aims to disperse infectious genetically modified viruses that have been
engineered to edit crop chromosomes directly in fields
 the means of delivery of these viral horizontal environmental genetic alteration
agents (HEGAAs) into the environment should be insect-based dispersion
 Part of scientific community does not find the program useful for the U.S.
agriculture, but points to its possible misuse
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Editing as a bioweapon?
Reeves et al., 2018
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 Medicine
 Molecular Diagnosis
 Personalized Medicine
 Gene Therapy
 Biotechnology
Outline
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BIOTECHNOLOGY
 It uses living organisms, cells or parts of cells (enzymes) for
research, leading to new products and applications in
medicine, agriculture, food, environmental protection
 Also used in developing better/sustainable production
methods for the chemical industry and other industrial
processes
 An interdisciplinary approach requiring knowledge of
chemistry, biology, physics, material sciences, engineering
and informatics
 The origin of biotechnology can be traced 4,000 years back,
when the Sumerians (although not knowingly) used microbes
for the production of alcoholic beverages.
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BIOTECHNOLOGY
 Examples
 effective utilization of plant biomass for fuel production
 acquisition of starting material (monomers) for the
production of polymers from living organisms instead of
from fossil sources
 phytopharmaceuticals – using plants in new vaccination
methods such as expression of antibodies or antigens
suitable for immunization
 European Federation of Biotechnology
http://www.efb-central.org
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 Medicine
 Molecular Diagnosis
 Personalized Medicine
 Gene Therapy
 Biotechnology
 Genetically Modified Organisms
 Transgenosis
Outline
31
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
1961 1997 2050
hectarespercapita
Source: UN Millennium Ecosystem Assessment
Human Population vs
Arable Land Availability
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Nutrition Deficiency
https://qz.com/africa/1064653/the-world-could-run-out-of-food-two-decades-earlier-than-thought/
…as soon as in 2027?
The world-total deficiency in food production of…
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 organisms naturally vary due to mutations
 before the era of genetic engineering question
of chance
 breeding tools
 selection and crossing
 modern breeder learned to change hereditary
information – increase the mutants allele
frequency
 chemicals, radiation ...
 results are incidental/non-targeted
Success
is not always visible at a glance
Breeding
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 Targeted modification ("targeted breeding")
 ability to transfer genes = transgenosis
 the first practical application: production of
human insulin in bacteria - 1978
Genetic Engineering
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Plant Transgenosis
https://www.youtube.com/watch?v=yesNHd9h8k0
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Breeding Vs. Genetic Engineering
37
 Organisms carrying modified genetic information –
either own or foreign (from another organism),
enabling targeted changes in the organism and its
use for specific purposes
 GMOs
 plants
 bacteria
 animals
http://www.gmo-compass.org/
Geneticaly Modified Organisms
(GMOs)
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 resistance to pests
 herbicide resistance
 resistance to drought
 resistance to cold
 resistance to salinity
 more efficient nitrogen utilization
 increasing nutritional quality
http://ipbo.vib-ugent.be/
Geneticaly Modified Plants
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 resistance to insect pests
 corn, cotton, rice
 genes from Bacillus thuringiensis
(Bt)
 Expression of crystalline deltaendotoxins
- Crystal (Cry) proteins
 increasing yields, reducing the
amount of chemical sprays
Bt Plants
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Bt Plants
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Ht Plants
 resistance to systemic herbicides
 glyphosate
 interferes with the synthesis of aromatic amino acids; animals
without the appropriate enzymatic apparatus = harmless
 blocks the enzyme 5-enolpyrovylshikimate-3-phosphate
synthase (EPSPS) in chloroplasts – affects green plants
 ineffective for bacterial EPSPS - evolutionarily divergent
 soya, maize, sugar beet, canola, cotton, alfalfa - added
enzyme for tolerance
 company Bayer (Monsanto), trade name Roundup
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Ht Plants
 resistance to systemic herbicides
 glufosinate (phosphinothricin)
 prevents processing of ammonium - toxic
 Streptomyces hygroscopicus synthesizes and transforms it:
acetylation by the enzyme phosphinothricin acetyltransferase
– coding gene isolated in 1987 - named bar
 trade names: Basta, Liberty, Finale, Radical ...
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Multiresistant Plants
 Bt resistance + herbicide
 multiresistant corn - the majority of total
production in the USA
 example of multiresistant corn:
 three Bt genes for resistance to air pests
 three Bt genes for resistance against soil pests
 two genes for herbicide resistance
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 viruses - no chemical agents available
 gene encoding non-infectious viral envelope protein increases
resistance to viral infection
 banana; papaya - Hawaii, Southeast Asia
 cassava - a basic food ingredient for more than 500 million people +
animal feed
Disease-Tollerant Plants
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 Chickpeas - more resistant to
drought, but toxic
 GMOs with inactivated toxin
 Corn resistant to drought
Lathyrus sativus
Chickpea
Cereals
Drought in Ethiopia
Disease- and Stress-Tollerant Plants
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 use of nitrogen from fertilizers
 rice with gene from barley - 3x higher nitrogen utilization
under oxygen deficiency
Nitrogen Use Efficiency
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 Golden rice
 several genes from maize encoding enzymes
for the biosynthesis of β-carotene (precursor of
vitamin A)
 Canola and Soybean
 improved oil properties: stable, resistant to high
temperatures, long storage
Improved Nutrition Value
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 Transgenic cats
 lentiviruses are sensitive to restriction factors
 specific restriction factor: rhesus macaque TRIMCyp + eGFP
 uniform expression, no mosaicity and no silencing in F1 generation
 lymphocytes of transgenic animals resistant to replication of FIV
Wongsrikeao et al., 2011, Nature Methods
GMO Animals
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 Medicine
 Molecular Diagnosis
 Personalized Medicine
 Gene Therapy
 Biotechnology
 Genetically Modified Organisms
 Transgenosis
 Genome Editing
Outline
50
Gene Editing in Plant
Domestication
51
 Medicine
 Molecular Diagnosis
 Personalized Medicine
 Gene Therapy
 Biotechnology
 Genetically Modified Organisms
 Transgenosis
 Genome Editing
 Model Organisms
Outline
52
 Low requirements for area
 Relatively large number of offspring (3-14, 6-8 on
average)
 Genome size is close to the size of human genome
(about 3000 Mbp), the number of genes as well (about
24K)
 20 chromosomes (19+1)
 Suitable for a wide range of physiological experiments
(anatomical and physiological similarity to human)
 Possibility to obtain (quite easily) KO mutants and
transgenic lines
Mus musculus
house mouse
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 Genome known since 2002
(http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/mouse/)
Mus musculus
house mouse
54
 Low requirements for cultivation area
Arabidopsis thaliana
mouse-ear cress
 High number of seeds (20.000 per plant and more)
 Small and compact genome, (125 MBp, about
25.000 genes, average size 3 kb)
 5 chromosomes
 Suitable for wide range od physiological experiments
 High natural variability (approximately 750 ecotypes
(Nottingham Arabidopsis Seed Stock Centre))
Columbia 0 Landsberg 0 Wassilewskija 0
http://seeds.nottingham.ac.uk/
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Arabidopsis thaliana
mouse-ear cress
 Genome known since 2000 (http://www.arabidopsis.org/)
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 Medicine
 Molecular Diagnosis
 Personalized Medicine
 Gene Therapy
 Biotechnology
 Genetically Modified Organisms
 Transgenosis
 Genome Editing
 Model Organisms
 Principles of PCR
Outline
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Discussion