Agricultural Biotechnology

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Agricultural Biotechnology

Flower colour modification via CRISPR‐Cas9 ribonucleoproteins

By : Karar, Ruchit, Rajvi, Jai

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Study Prospect - Yu, J., Tu, L., Subburaj , S.  et al.  Simultaneous targeting of duplicated genes in  Petunia protoplasts for flower color modification via CRISPR-Cas9 ribonucleoproteins.

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Background to the Research

Agricultural biotechnology : CRISPR -CAS9 DNA sequence found in the genomes of prokaryotic organisms such as bacteria. These sequences are derived from DNA fragments of bacteriophages that had previously infected the prokaryote. used to detect and destroy DNA from similar bacteriophages during subsequent infections. CAS9 protein (Endonuclease) is a four-component system that includes two RNA molecules :crRNA, tracrRNA.

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Background to the Research

CRISPR-CAS9: crRNA and tracrRNA are fused together into a "single-guide RNA" that, when combined with Cas9, could find and cut the DNA target specified by the guide RNA. In plants: Agrobacterium tumefaciens -mediated transformation system is most popular method for CRISPR-mediated genome editing to deliver DNA encoding the components Issues with this method: T-DNA integration in the host plant genome Frequently inducing unwanted genetic alterations Constitutive expression of CRISPR-Cas9 complexes Which can increase off-target mutations in the genome

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Motivation

Cas9-ribonucleoproteins (RNPs) delivery (Hempstead n.d. 2021) Performed site-specific mutagenesis in Petunia to engineer flower colour modifications Has many advantages in plants: DNA-free genome editing Does not require codon optimization It can be used directly in various crop species Cleave target DNAs immediately after transfection Rapidly degraded in cells Reduces the number of unwanted mutations at off-target sites

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Hypothesis/Aim

Flower colour is an important trait for ornamental plants like Petunia because it has a strong influence on their market value. Flower colours in petunia are determined by genes that are involved in the flavonoid/anthocyanin biosynthesis pathway F3H is one of the key enzymes in this pathway F3H catalyses the 3-hydroxylation of (2S)-flavanones such as naringenin to dihydroflavanols, which are important for the synthesis of anthocyanidin and flavanols pigments. Many studies have aimed to modify flower colour by inhibiting or over-expressing the F3H gene Therefore, in this study, they selected F3H as a target for editing by the CRISPR-Cas9- (RNPs) system to change Petunia colour. Flower color is an important trait for ornamental plants like Petunia because it has a strong influence on their market value.

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An outline for Crispr Cas9

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This Photo by Unknown Author is licensed under CC BY

This Photo by Unknown Author is licensed under CC BY

This Photo by Unknown Author is licensed under CC BY-NC-ND

This Photo by Unknown Author is licensed under CC BY-NC-ND

Audio Recording 13-Sep-2021 at 12:45:18 AM

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https://www.yourgenome.org/facts/what-is-crispr-cas9

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How does it work?

Audio Recording 13-Sep-2021 at 12:54:51 AM

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Design of guide RNAs that target two F3H genes at once in Petunia selective breeding Sanger sequencing Transformation NGS

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This Photo by Unknown Author is licensed under CC BY

This Photo by Unknown Author is licensed under CC BY

Audio Recording 13-Sep-2021 at 1:07:35 AM

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Other Gene-editing technologies

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This Photo by Unknown Author is licensed under CC BY

This Photo by Unknown Author is licensed under CC BY

Audio Recording 13-Sep-2021 at 1:26:48 AM

TALENs ZFNs

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Other Applications of The RNP Method of CRISPR

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CRISPR is a Very Versatile Biotechnology

CRISPR/Cas9

Gene Editing

Gene Libraries for Screening

Transcriptional Activation/Repression

Epigenetic Editing

Live Imaging of DNA/mRNA

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Agricultural Applications

“We demonstrated a precedent of ornamental crop engineering by a DNA-free CRISPR method for the first time, which will greatly accelerate a transition from laboratory to fields.” Paved the way for transgene free plants and crops . Some examples include browning-resistant mushrooms, high-amylopectin waxy corn, and false flax with enhanced omega-3 oil. These were all developed using CRISPR and approved by the US Department of Agriculture (USDA) (Zaidi, Mahas, Vanderschuren and Mahfouz, 2020). Research groups have demonstrated genome editing via Cas9-RNPs in Arabidopsis, rice, lettuce, tobacco, microalgae, grape, apple, and potato. Ability to make crops more resistant to combat food insecurity.

A CRISPR Mushroom — Penn State College of Agricultural Sciences

Advanced breeding technology has promising future in agriculture – Ohio Ag Net | Ohio's Country Journal

Anti Browning Mushrooms

High-Amylopectin Waxy Corn

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Medical Applications

This technology has applications in treating genetic diseases caused by point mutations CRISPR Cas 9 RNP method has recently (2020) been used in a trial to help people with sickle cell anaemia and beta-thalassemia(Prabhune, 2021). mutation in the β-haemoglobin gene, which encodes one of the two subunits of adult haemoglobin (HbA, α 2 β 2 ). CRISPR was used by transferring the RNP complex, RNA guide and Cas 9 proteins into haemopoietic and progenitor stem cells (Yen et al., 2018). Modified cells reinserted back into patients with promising results. Similar trials being used for people with aids and blindness.

CRISPR CTX001 Clinical Trial: Promising Results in Sickle Cell Patients

CRISPR/Cas9 genome engineering in hematopoietic cells - ScienceDirect

Example Procedure: Ex vivo

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Future Applications

Creating new biofuels to reduce greenhouse gas emissions through modifying algae and bacteria especially the Clostridium autoethanogenum bacterium (Nagaraju et al., 2016). T cell modifications for cancer treatment (CAR-T cell) (Bryant, 2021). Another future application is the controversial one of extending natural life span through the modification of stem cells (Zhang et al., 2017). Shown to be a correlation between hypothalamic stem cells and ageing in mice.

Improved Safety in CAR T Cell Therapy | Taconic Biosciences

CAR-T Cell Formation and Method

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Safety and ethical concerns

Plant Biotechnology-environmental and biosafety concerns Ethical implications of CRISPR-Cas9 Human health safety concerns

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Plant biotechnology -environmental and bio safety concerns

Off target effects: On crossing different plants, oftentimes, the progeny plants end up having undesirable characteristics. They are usually discarded into the environment and other species of organisms including animals could be harmed. It could cause accidental gene flow and give rise to unwanted plant types which might include superweeds. Concern for other organisms: Plants and crops are widely consumed by different organisms in general. This typically involves herbivores and a certain population of humans. Genetically modified plants are found to produce toxins and they could be harmful for other species apart from their own. Genetic erosion and biodiversity loss: It is beneficial to have biotechnological techniques to tweak the natural plants and their genomes. However, it might lead to genetic erosion and greatly affect biodiversity in a longer run as wildtype strains are overlooked. (Toms B, 2013; Louwaars N and Jochemsen H, 2021) Such interventions cause harmful adversities and might not remain confined to the plant species involved but could thus propagate to environment.

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Ethical considerations of CRISPR-Cas9

Consequentialist approach

Naturalness

Science based naturalness

This approach measures the efficacy of human innovations in terms of risks and benefits. It suggests that the risks run equally in both non-GMO and GMO plants. Natural evolution could bring out similar kinds of genetic modifications as GMO plants. Thus, it could be concluded that CRISPR-Cas9 poses fewer risks than the conventional GM techniques.

Naturalness of breeding in plants could be viewed as a complex process that cannot be hindered by humans. However, with increasing demands and purposeful experimentations, any biotechnological technique could prove to be an intrusion in the natural genetic pool of plants. Thus, making it less morally acceptable as nature is disturbed.

Biotechnology is a field which mimics natural processes. In that manner, it is believed that genetic modification of plants is as natural as nature itself. Further, the edition or transfer of genes in the lab is compared to biological natural processes and concluded that it is all morally acceptable but does not advocate the same for humans.

( Bartkowski B et al, 2018)

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Ethical and safety issues concerning humans

Potential for exhibiting toxicity and allergies: Introduction of new genes in a plant could mean new characteristics on exhibit. Some of them could be harmful for humans. For example, genetically modified DNA from food crops affects the gut microbiome of human which could be raising different health issues. Many could trigger allergies which might take a severe form in humans with chronic susceptibility to allergies. Social Justice: GM technology not only has a direct effect on plants and growers but also on multinational companies that invest in such technology and its products. The farmers’ Rights are being taken over by the business and tech owners which violates their filed pf occupation and their rights over their produce. (Toms B, 2013; Louwaars N and Jochemsen H, 2021)

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In depth analysis Informed decisions Rights from wrongs Facts over controversies and myths

HOW TO DEAL WITH SUCH ISSUES?

Harfouche A et al, 2021

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References

Yu, J., Tu, L., Subburaj , S.  et al.  Simultaneous targeting of duplicated genes in  Petunia protoplasts for flower color modification via CRISPR-Cas9 ribonucleoproteins.  Plant Cell Rep   40,  1037–1045 (2021). https://doi.org/10.1007/s00299-020-02593-1 Hempstead, A n.d., CRISPR 101: Ribonucleoprotein (RNP) Delivery , viewed 12 September 2021, <https:// blog.addgene.org /crispr-101-ribonucleoprotein-rnp-delivery>. What is Crispr Cas9? https://www.yourgenome.org/facts/what-is-crispr-cas9 Zaidi, S., Mahas , A., Vanderschuren , H. and Mahfouz, M., 2020. Engineering crops of the future: CRISPR approaches to develop climate-resilient and disease-resistant plants.  Genome Biology , [online] 21(1). Genscript.com , 2021, Applications of CRISPR [online], Available at: https://www.genscript.com/applications-of-crispr.html , Accessed 10 September 2021. Prabhune , M., 2021.  Synthego Full Stack Genome Engineering . [online] Synthego.com . Available at: https://www.synthego.com/blog/Crispr-applications , Accessed 10 September 2021. Yen, J., Fiorino, M., Liu, Y., Paula, S., Clarkson, S., Quinn, L., Tschantz , W., Klock , H., Guo, N., Russ, C., Yu, V., Mickanin , C., Stevenson, S., Lee, C. and Yang, Y., 2018.,TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells.  Scientific Reports , [online] 8(1). Zhang, Y., Kim, M., Jia, B., Yan, J., Zuniga-Hertz, J., Han, C. and Cai, D., 2017. Hypothalamic stem cells control ageing speed partly through exosomal miRNAs, Nature , [online] 548(7665), pp.52-57, Nagaraju , S., Davies, N., Walker, D., Köpke , M. and Simpson, S., 2016, Genome editing of Clostridium autoethanogenum using CRISPR/Cas9,  Biotechnology for Biofuels , [online] 9(1), Available at : https:// biotechnologyforbiofuels.biomedcentral.com /articles/10.1186/s13068-016-0638-3 , [Accessed 12 September 2021 .

Yu, J., Tu, L., Subburaj , S.  et al.  Simultaneous targeting of duplicated genes in  Petunia protoplasts for flower color modification via CRISPR-Cas9 ribonucleoproteins.  Plant Cell Rep   40,  1037–1045 (2021). https://doi.org/10.1007/s00299-020-02593-1 Hempstead, A n.d., CRISPR 101: Ribonucleoprotein (RNP) Delivery , viewed 12 September 2021, <https:// blog.addgene.org /crispr-101-ribonucleoprotein-rnp-delivery>. What is Crispr Cas9? https://www.yourgenome.org/facts/what-is-crispr-cas9 Zaidi, S., Mahas , A., Vanderschuren , H. and Mahfouz, M., 2020. Engineering crops of the future: CRISPR approaches to develop climate-resilient and disease-resistant plants.  Genome Biology , [online] 21(1). Genscript.com , 2021, Applications of CRISPR [online], Available at: https://www.genscript.com/applications-of-crispr.html , Accessed 10 September 2021. Prabhune , M., 2021.  Synthego Full Stack Genome Engineering . [online] Synthego.com . Available at: https://www.synthego.com/blog/Crispr-applications , Accessed 10 September 2021. Yen, J., Fiorino, M., Liu, Y., Paula, S., Clarkson, S., Quinn, L., Tschantz , W., Klock , H., Guo, N., Russ, C., Yu, V., Mickanin , C., Stevenson, S., Lee, C. and Yang, Y., 2018.,TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells.  Scientific Reports , [online] 8(1). Zhang, Y., Kim, M., Jia, B., Yan, J., Zuniga-Hertz, J., Han, C. and Cai, D., 2017. Hypothalamic stem cells control ageing speed partly through exosomal miRNAs, Nature , [online] 548(7665), pp.52-57, Nagaraju , S., Davies, N., Walker, D., Köpke , M. and Simpson, S., 2016, Genome editing of Clostridium autoethanogenum using CRISPR/Cas9,  Biotechnology for Biofuels , [online] 9(1), Available at : https:// biotechnologyforbiofuels.biomedcentral.com /articles/10.1186/s13068-016-0638-3 , [Accessed 12 September 2021 .

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References

Bryant, K., 2021, Synthego Full Stack Genome Engineering, [online] CAR T Therapies and CRISPR are Fighting Cancer and Revolutionizing Medicine, Available at: https://www.synthego.com/blog/car-t-Crispr-cancer , Accessed 10 September 2021. Toms, B. (2013). Ethical Concerns in Plant Biotechnological Research. International Journal of Biotechnology and Bioengineering Research, [online] 4(3), pp.197–204. Louwaars, N. and Jochemsen, H. (2021). An Ethical and Societal Analysis for Biotechnological Methods in Plant Breeding. Agronomy, [online] 11(6), p.1183. ‌‌ Bartkowski , B., Theesfeld , I., Pirscher , F. and Timaeus, J. (2018). Snipping around for food: Economic, ethical and policy implications of CRISPR/Cas genome editing. Geoforum , [online] 96, pp.172–180. Images Penn State College of Agricultural Science. 2021.  A CRISPR Mushroom . [online] Available at: https://agsci.psu.edu/magazine/articles/2016/fall-winter/a-Crispr-mushroom , Accessed 10 September 2021. Ohio Ag Net Country Journal, 2021, Advanced breeding technology has promising future in agriculture, Available at: https://ocj.com/2017/06/advanced-breeding-technology-has-promising-future-in-agriculture/ , Accessed 10 September 2021. Prabhune , M., 2021.  Synthego Full Stack Genome Engineering . [online] Synthego.com . Available at: https://www.synthego.com/blog/Crispr-applications , Accessed 10 September 2021. Sürün , D., von Melchner , H. and Schnütgen , F., 2018, CRISPR/Cas9 genome engineering in hematopoietic cells,  Drug Discovery Today: Technologies , [online] 28, pp.33-39, Biosciences, T., 2021,  Improved Safety in CAR T Cell Therapy, [online] Taconic.com , Available at: https:// www.taconic.com / taconic -insights/oncology-immuno-oncology/car-t-cell- safety.html , Accessed 10 September 2021. What is Crispr Cas9, September 2020 https://www.yourgenome.org/facts/what-is-crispr-cas9.html https:// edelweisspublications.com /keyword/27/783/Plant-genetics

Bryant, K., 2021, Synthego Full Stack Genome Engineering, [online] CAR T Therapies and CRISPR are Fighting Cancer and Revolutionizing Medicine, Available at: https://www.synthego.com/blog/car-t-Crispr-cancer , Accessed 10 September 2021. Toms, B. (2013). Ethical Concerns in Plant Biotechnological Research. International Journal of Biotechnology and Bioengineering Research, [online] 4(3), pp.197–204. Louwaars, N. and Jochemsen, H. (2021). An Ethical and Societal Analysis for Biotechnological Methods in Plant Breeding. Agronomy, [online] 11(6), p.1183. ‌‌ Bartkowski , B., Theesfeld , I., Pirscher , F. and Timaeus, J. (2018). Snipping around for food: Economic, ethical and policy implications of CRISPR/Cas genome editing. Geoforum , [online] 96, pp.172–180. Images Penn State College of Agricultural Science. 2021.  A CRISPR Mushroom . [online] Available at: https://agsci.psu.edu/magazine/articles/2016/fall-winter/a-Crispr-mushroom , Accessed 10 September 2021. Ohio Ag Net Country Journal, 2021, Advanced breeding technology has promising future in agriculture, Available at: https://ocj.com/2017/06/advanced-breeding-technology-has-promising-future-in-agriculture/ , Accessed 10 September 2021. Prabhune , M., 2021.  Synthego Full Stack Genome Engineering . [online] Synthego.com . Available at: https://www.synthego.com/blog/Crispr-applications , Accessed 10 September 2021. Sürün , D., von Melchner , H. and Schnütgen , F., 2018, CRISPR/Cas9 genome engineering in hematopoietic cells,  Drug Discovery Today: Technologies , [online] 28, pp.33-39, Biosciences, T., 2021,  Improved Safety in CAR T Cell Therapy, [online] Taconic.com , Available at: https:// www.taconic.com / taconic -insights/oncology-immuno-oncology/car-t-cell- safety.html , Accessed 10 September 2021. What is Crispr Cas9, September 2020 https://www.yourgenome.org/facts/what-is-crispr-cas9.html https:// edelweisspublications.com /keyword/27/783/Plant-genetics