Part 1 Making plants resistant to viruses by 'transplanting' the bacterial CRISPR/Cas immune system to the plant

Various 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) 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).

Search for which parts. Identify all components of the T-DNA in Figure 1A, and give the function for each component.

• To get started: LB = left border, this is the left border of the T-DNA in A. tumefaciens.

• Even more help: NOS and Ter are 'terminators'. What is the function of a terminator?

7. 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. Explain the lower part of Fig. 1A. What is pictured here?

9. What is shown in Fig. 1B?

10. And what in Fig. 1C?

Read the sections "Screening of the sgRNA-FnCas9 target sites" and "Establishing resistance in tobacco"

11. In Fig. 1d, e, 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. Read the section "Establishing stable resistance in Arabidopsis", and read Figure 5. In 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, and d?

    d. T2 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?