Research

Project Description

Our IMPACT-system contains cells that are capable of detecting phage infections. If our cells are infected with a certain phage they will either up-regulate or downregulate a reporter gene, e.g. GFP, leading to an easily detectable signal. In this section we will explain how we want to get this system to work.

In figure 1 (make something like poster here) a general overview of the system is given. In the first step a CRISPR complex will take up spacers from the invading phage DNA. In the following step the CrRNA, corresponding to the new spacer, is used by dCas, which is not part of the CRISPR-complex, to interfere with the expression of a reporter construct. We will target our dCas9 to the region between the transcription start site and the RBS by inserting a sequence similar to the spacer that is taken up from the phage. To determine which spacer this is we have designed a model, which will be discussed on the next page.

Finally the entire system needs to be constructed in L. lactis, which is a Lactic Acid Bacteria. Working with lactis requires some special attention, it is not as easily transformed as E. coli or B. subtilis and it generally E. coli or B. subtilis promotors don’t work in lactis. Moreover L. lactis is an important organism in fermentation industries, but not a lot of informations or parts can be found in the iGEM database. Therefore we created a fourth sub-project, called lactis-toolbox, in which we share the problems we encountered, our protocols and we have created three new lactis promotor parts. Since our entire project involves around CRISPR-Cas we will start by giving a short summary of all important things that you need to know about CRISPR-Cas before going into more detail on the different sub-projects.

In our project we will make use of the CRISPR-Cas system of Streptococcus pyogenes. This system is a type II-A and it consists of four proteins and two RNA molecules [Wei 2015)]. The natural CRISPR-system is a defence mechanism against invading bacteriophages (or DNA?). Often only one part of the system, namely Cas9, is used to target specific sequences in the genome. Next a quick overview of the mechanism behind CRISPR immunity will be given followed by a more detailed description of all components.

CRISPR immunity can be seen as a three stage process (Figure …) [http://rna.berkeley.edu/crispr.html]. The first stage starts after a phage has infected the cell with its DNA. From this DNA a spacer is acquired, this is a small (20 nt) fragment originating from the infectious DNA. This spacer is built into the CRISPR array, which is a collection of multiple spacers flanked by repeat regions. In the second stage of defence, this array is transcribed and a pre-CrRNA is produced, which is subsequently processed into separate CrRNA molecules. Finally Cas9 can use the processed Cr-RNA’s together with a TracrRNA to target a specific sequence encoded by the spacer that was taken up in the first stage. Since the CRISPR array is (relatively?) stable the cell will have a memory of the infection and will be protected against future infections of similar phages.

The four proteins (Cas1, Cas2, Csn2 and Cas9) are expressed together in one operon, which in the natural system also contains the TracrRNA. In our project we will use a plasmid, constructed by [Heler?] containing only the four proteins coding genes. Therefore all references to the CRISPR operon will be referring to the organization on this plasmid and not the natural organization.

The TracrRNA is one of the two RNA molecules in the natural CRISPR system. This part directs the second RNA molecule to the Cas9 protein. This second RNA molecule is called the CRISPR-RNA (CrRNA) and contains a repetitive region, which binds to the TracrRNA, and a sequence targeting part.

The CrRNA is transcribed from the CRISPR-array, which can be seen as the memory of the CRISPR-system. In this array all spacers taken up from invading DNA sequences are built in between two repeats. Therefore during an infection the array will grow in size as spacers are taken up and they are together with an extra repeat incorporated into the array. The transcript of the array (pre-CrRNA) is processed into individual CrRNA’s consisting of one repeat and one spacer. This CrRNA’s are together with the TracrRNA used by Cas9 to target sequences identical to spacer.

The first sub-project is concerned with the spacer acquisition for which we use a slightly adapted version of the S. pyogenes CRISPR-Cas system. Instead of using the normal CRISPR system we will use one with an hyperactive Cas9 (hCas9). Heler et al. described a single amino acid mutation that could turn Cas9 into hCas9, which resulted in an ~100 fold increase of the spacer uptake rate [Heler (2017)].

We have chosen for the hCas9-variant, because in general the spacer uptake rate of CRISPR-Cas is not very high and therefore the chance that the spacer for which we pre-programmed the cells is take up is also quite low. With the use of the hyperactive

A disadvantage of the hyperactive Cas9 is that it is also known to take up spacers from plasmid or even the genome. When hCas9 is directed to these parts this will result in clearing of the plasmid and the corresponding antibiotic marker, which will lead to cell death. Therefore the hyperactive CRISPR array might have a severe fitness effect on the cells, but because we will use a second Cas9 variant for CRISPR interference it should not interfere with the detection process.

For the CRIPR-interference part we have decided to use a second specialised Cas9 variant, namely dCas9 with a changed PAM preference. Both Cas9s can be directed by the same spacers as their guides use the same crRNA and tracrRNA structure. However, four amino acid substitutions [table …] in Cas9 changes its PAM preference to NGCG [Kleinsteiver et al.]. Our plan is to combine the PAM-altering mutations with those of dCas9 to obtain a dCas9VRER. Using this dCas9 with an altered PAM preference afforded us a great number of benefits.

First of all, spacers taken up by hCas9 need to be flanked by an NGG on the original sequence. In our target array the targeted spacers are flanked by NGCG and therefore our target array won’t be targeted by the hCas9. Thus our plasmid won’t be cut and the cells will stay resistant against the antibiotic.

Next since the hCas9 is so much more efficient in taking up spacers than dCas9, it will be very unlikely that dCas9 will take up a spacer, which would be flanked by an NGCG sequence. As a result it is not likely that our detector will give off a signal after a spacer from the plasmid is taken up. If hCas9 would take up a spacer from the plasmid the cells will die, as discussed in the previous section.

Combining the two Cas9 variants is something that to our knowledge has never been done and unfortunately we have not succeeded in combing the two proteins in our project. However we did succeed in combining the dCas9 mutations and the PAM altering mutations. … something results?

A fourth sub-project that is not involved with the project directly is the lactis-toolbox. Since we want to make our product suitable for the dairy industry we need to incorporate all our parts into L. lactis in the end. However parts that work in E. coli do often not work in L. lactis directly, promotors for example often need to be interchanged also it requires specific cloning protocols. Therefore we have uploaded several protocols, which we obtained from our supervisor Patricia, and we have tested and validated three L. lactis promotors.

1. Kleinstiver, B. P. et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015).
2. Heler, R. et al. Mutations in Cas9 Enhance the Rate of Acquisition of Viral Spacer Sequences during the CRISPR-Cas Immune Response. Mol. Cell 65, 168–175 (2017).
3. Larson et al. CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nature Protocols, 8(11), pp.2180-2196. (2013).
4. Wei, Y., Terns, R. M., & Terns, M. P. (2015). Cas9 function and host genome sampling in Type II-A CRISPR–Cas adaptation. Genes & Development, 29(4), 356–361.