Planten resistent maken tegen virussen door ‘transplantatie’ van het bacteriële CRISPR-Cas afweersysteem naar de plant
Verschillende bacteriesoorten hebben immuunsystemen ontwikkeld tegen virussen. Eén immuunsysteem heet CRISPR-Cas. De bacterie knipt met CRISPR-Cas het DNA van virussen die de bacterie binnendringenVarious bacterial species have developed immune systems against viruses. One immune system is called CRISPR/Cas. The bacterium uses CRISPR/Cas to cut the DNA of viruses that enter the bacterium. Zhang et al. (2018) (https://doi.org/10.1111/pbi.12881) hebben zo’n CRISPR-Cas systeem, afkomstig uit een bacterie, in de plant gezet, met als doel planten resistent te maken tegen virussen. Het bacteriële immuunsysteem is als het ware getransplanteerd naar de plant.
- Lees het abstract van dit artikel zorgvuldig door.
- Lees ook de Introduction.
- Lees de eerste paragraaf van de resultaten
- Bestudeer figuur 1A (Pagina 1417)
en beantwoord dan de volgende vragen:
- Wat is het doel van dit onderzoek?
- Moet CRISPR-Cas hier een plantengen knippen of een virusgen?
- Beschrijf het principe van de resistentie. Hoe werkt het?
- Typen virussen
- Voor welke twee virussoorten is de resistentie ontwikkeld?
- Zoek (online) op wat voor typen virussen er allemaal zijn volgens de Baltimore classificatie.
- Tot welke groep van Baltimore behoren de twee virussen uit vraag 4a?
- In dit artikel wordt gesproken over RNA-virussen, oftewel virussen die geen DNA hebben, maar RNA. Cas9 knipt normaal gesproken in DNA, maar niet in RNA! Hoe hebben de auteurs dit opgelost? Lees hierover het einde van de Introduction en de eerste paragraaf van de Discussion.
- CRISPR-Cas bestaat uit 2 onderdelen: Het CRISPR-deel en het Cas-deel. Het Cas-gen codeert voor het Cas eiwit. Het Cas-eiwit knipt in DNA of RNA. De plaats waar die knip wordt aangebracht wordt bepaald door de single stranded guide RNA (sgRNA, ook wel gRNA genoemd) in het CRISPR-deel.
- Leg uit hoe dit werkt. Als je wilt, kun je bij je uitleg verwijzen naar het onderste deel van Fig. 1A.
- Ik zei net “De plaats waar die knip wordt aangebracht wordt bepaald door de sgRNAsgRNAs in het CRISPR-deel.” Dat klopt, maar er is een element dat de specificiteit nog verder verhoogt. Welk element?
- Hoe wordt de specificiteit bereikt van het CRISPR/Cas9 systeem, waardoor het tegen specifieke virussen werkt, maar niet tegen andere virussen? Knipt het ook in planten-RNA?
Results. Figuur 1.
Figuur 1A laat de opbouw zien van het T-DNA dat de onderzoekers hebben gebouwd, en in een vector hebben gezet. Die vector hebben ze in Agrobacterium tumefaciens gebracht, en vervolgens heeft A. tumefaciens het stukje DNA tussen de left border (LB) en right border (RB) in de plant gebracht. Het stukje DNA tussen deze twee borders heet have placed such a CRISPR/Cas system, originating from a bacterium, in a plant, aiming at making plants resistant to viruses. The bacterial immune system is, as it were, transplanted to the plant.
Read the abstract of this article carefully.
Also read the Introduction.
Read the first paragraph of Results
Study Figure 1A (Page 1417) and then answer the following questions:
1. What is the purpose of this research?
2. Should CRISPR/Cas cut a plant gene or a virus gene here?
3. Describe the principle of resistance. How does it work?
4. Types of viruses
a. For which two types of virus has the resistance been developed?
b. Search (online) for what types of viruses exist according to the Baltimore classification.
c. Which Baltimore group are the two viruses from question 4a?
d. This article talks about RNA viruses, i.e., viruses that do not have DNA, but RNA. Cas9 normally cuts DNA, but not RNA! How did the authors solve this? Read about this at the end of the Introduction and the first paragraph of the Discussion.
5. CRISPR/Cas consists of two parts: the CRISPR part and the Cas part. The Cas gene codes for the Cas protein. The Cas protein cuts into DNA or RNA. The place where the cut is made is determined by the single stranded guide RNA (sgRNA, also known as gRNA) in the CRISPR part.
a. Explain how this works. If you want, you can refer to the lower part of fig. 1A.
b. I just said "The place where that cut is made is determined by the sgRNAs in the CRISPR part." That's correct, but there is an element that increases the specificity even further. Which element?
c. How is the specificity of the CRISPR/Cas9 system achieved, which makes it work against specific viruses, but not against other viruses? Does it also cut in plant RNA?
Results. Figure 1.
6. Figure 1A shows the structure of the T-DNA that the researchers have built and put into a vector. They brought that vector into the bacterium Agrobacterium tumefaciens, and then A. tumefaciens introduced the piece of DNA between the left border (LB) and right border (RB) into the plant. The piece of DNA between these two borders is called T-DNA (transfer DNA).
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Search for which parts. Identify all components of the T-DNA in
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Figure 1A,
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and give the function for each component.
To get started: LB = left border,
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this is the left border of the T-
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DNA in A. tumefaciens.
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Even more help: NOS and Ter are 'terminators'. What is the function of a terminator?
7.
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From which bacteria is the Cas gene used here? Why was this bacterium chosen, and not a much more common bacterium, such as Streptococcus?
8.
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Explain the lower part of Fig. 1A
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.
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What is
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pictured here?
9.
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What is shown in Fig. 1B
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?
10.
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And what in Fig. 1C?
Lees de paragrafen “Read the sections "Screening of the sgRNA–FnCas9 sgRNA-FnCas9 target sites” en “" and "Establishing resistance in tobacco”"
11. In Fig. 1d, e,
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Figuur and f the results are shown. What is meant by Mock vector? And what do the other vectors contain? What do the author want to show with these bar charts?
Figure 5.
12.
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Read the section "Establishing stable resistance in Arabidopsis", and read Figure 5. In
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Figure 1, the researchers were still working on a transient test. That is, T-DNA was infiltrated into leaf, and was active for a while at that location. But no transgenic plants were made in which each cell contained the T-DNA. This is different in Figure 5. Transgenic plants have been made, in which the T-DNA is stably incorporated in the DNA of each cell of the plant.
a. In Figure 5A, 9 plants are shown. What do the authors mean by the 3 codes at the top row of 3 plants? Tip: a small letter "p" indicates that it is a name of a vector (p of plasmid).
b. Which plants in Fig. 5A have a CRISPR/Cas construct against the CMV virus, and which plants do not have this construct? What do you notice in the plants with a CRISPR/Cas construct compared to the plants without the construct?
c. What do the authors want to show with Fig. 5 b, c,
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and d?
d. T2
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plants are plants from the self-pollination of a transgenic mother plant (T1). Will a transgenic mother plant be homozygous or heterozygous for the T-DNA insertion?
e. If you apply self-pollination to a mother plant with 1 T-DNA insertion, how many percent of the T2 offspring will not contain the transgene, and how many percent do the transgenes do?
f. How many percent of the T2 offspring would be resistant? Do you expect a difference in resistance level between offspring that are homozygous for the insertion and offspring that are heterozygous for the insertion?