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Flower colour modification via CRISPR‐Cas9 ribonucleoproteins

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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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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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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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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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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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This Photo  by Unknown Author is licensed under  CC BY

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

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

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Other Applications of The RNP Method of CRISPR

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“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&#39;s Country Journal

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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

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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

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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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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.

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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

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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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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