A New Revolution with CRISPR/Cas9?

MBAA Blog 2 - CRISPR/Cas9 

1. In your own words, but using the correct scientific terms, explain the involved mechanisms of the system (in more details as in the lecture).

Genome engineering has been around for a while as an alternative to traditional breeding strategies to improve traits of crop plants (Belhaj 2013). With CRISPR/Cas9 it’s possible to make targeted modifications to the genome in any organism of choice. Editing sequences of DNA will allow researchers to better understand the functional organisation of the genome and the causal genetic variations (Hsu et al., 2014). CRISPR is easier and more efficient than previous genome engineering methods (ZFNs or TALENs) (Kumar et al., 2015).

CRISPR was first discovered in E. coli as a kind of immune system against viral DNA. Upon viral DNA insertion into a bacterial cell, a part of the viral DNA is cut and inserted at a specific location (CRISPR sequence) in the bacterial DNA. This sequence is then translated by the cell itself into a CRISPR-RNA molecule containing genetic information of both bacteria and virus. Another molecule, the tracer RNA, joins in. Together, these two molecules (guide-RNA) bind to a protein called Cas9. The guide-RNA can bind to the inserted viral DNA whereupon the Cas9 enzyme cuts the DNA strands rendering the virus harmless. The CRISPR-RNA determines where Cas9 cuts DNA. As tracer and CRISPER-RNA can be combined into one single molecule the production has become much easier. (Max-Planck-Gesellschaft, 2015, youtube)

Slightly modified Cas9 work also in cells with nucleus of higher organisms. As the sequence of the fused RNA molecule can be varied, researchers can determine where Cas9 cuts DNA. The cell tries to repair the broken strands but succeeds only partially. Mutations occur and the gene can no longer be transcribed. By cutting at a specific location genes can be switched off. Besides the inactivation of a gene, the complete exchange of genes is also possible by simply inserting a donor DNA. Understanding this process holds tremendous potential in various scientific fields. The CRISPR method could also be applied in plant biotechnology to engineer plants with specific traits by inserting the desired genes at specific loci. The identification of a gene location and precise cutting of the DNA has the potential to even heal hereditary diseases. (Max-Planck-Gesellschaft, 2015, youtube), (Paul Andersen, 2016, youtube)

2. Give an example of the application of this system and its usefulness.

With climate change and an ever increasing world population the need for adaptable crops has becomes apparent. With CRISPR/Cas9 genes of crop plants could be edited by adding or deleting desired or unwanted traits to a plant. Such traits might be a fungal resistance, the ability to grow in a salty environment or in a very dry environment and still produce enough yield. If a plant is resistant to a fungi, less fungicides might be necessary. With traditional breeding methods these goals might not be achievable in time. In order to cope with future agricultural challenges CRISPR/Cas9 could serve as an alternative method to improve crop plants.

3. What are the (technical) requirements for this method?

As mentioned above, CRISPR/Cas9 originally comes from bacteria where all required elements are present in the cell. When applied in plants, guide-RNA and the Cas9 protein need to be transferred into the living target cells. Kumar and Jain explain in their paper: “the sgRNA and Cas9 can be expressed in plant cells either from expression vectors or by microinjected RNA (for sgRNA) and mRNA (for Cas9)”. According to the authors, this can be done either directly by electroporation or via plasmids. Shotgun, agrobacterium-mediated transformation and polyethylene glycol-mediated transformation are methods that were applied to deliver the guide RNA (sgRNA) and Cas9 protein into the cells. Further, optimal promoters are imperative for an efficient genome editing. (Kumar and Jain, 2015), (Ann Ran et al., 2013)

4. In your opinion, are there any disadvantages on the use of this technology that would prevent its use for a large number of crops? If so, explain what could be problematic.

Whenever technologies are applied that involve genetically modified or genetically edited organisms or plants, ethical questions arise. The advantages and disadvantages must be discussed and weighed. After all, CRISPR/Cas9 is a very powerful tool which could do a lot of damage in the wrong hands. As far as I have understood, CRISPR is an efficient method which can be used to edit crop plants to add desired traits. If applied wisely, this technique could have advantages over traditional plant breeding. In my opinion, CRISPR/Cas9 needs to be regulated by an authority that neither represents big corporations and acts in their favour nor pursues any own goals for profit. Decisions must be made solely on the base of knowledge, ethics and research. Aspects like public health, responsibility for future generations and biodiversity must be taken into consideration. Moreover, we have to be sure to fully understand any consequences that may result from the use and/or misuse of this technology.

References

F Ann Ran^1–5,8, Patrick D Hsu^1–5,8,, Jason Wright1, Vineeta Agarwala^1,6,7, David A Scott^1–4 & Feng Zhang^1–4. (2013). Genome engineering using the CRISPR-Cas9 system. 2308 | VOL.8 NO.11 | 2013 | nature protocols

Khaoula Belhaj†, Angela Chaparro-Garcia†, Sophien Kamoun* and Vladimir Nekrasov*. (2013). Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system20. 

Patrick D. Hsu,^1,2,3 Eric S. Lander,¹ and Feng Zhang^1,2,*. (2014). Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell 157, June 5, 2014 ª2014 Elsevier Inc.

Vinay Kumar and Mukesh Jain. (2015). The CRISPR–Cas system for plant genome editing: advances and opportunities. Journal of Experimental Botany, Vol. 66, No. 1 pp. 47–57, 2015

Youtube films

How CRISPR lets us edit our DNA | Jennifer Doudna. (2015). Abgerufen am 05.04.2017 von https://www.youtube.com/watch?v=TdBAHexVYzc

CRISPR/Cas9 – ein Skalpell für das Erbgut. Max-Planck-Gesellschaft. (2015). Abgerufen am 05.04.2017 von https://www.youtube.com/watch?v=ouXrsr7U8WI

What’s CRISPR? Paul Andersen, Bozeman Science. (2016). Abgerufen am 05.04.2017 von https://www.youtube.com/watch?v=MnYppmstxIs