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.

Subsequently the signal, the spacer, will have to be detected. This task will be performed by a second Cas9 variant, namely dCas9. The catalytically dead Cas9, which is a result of two mutations (table), cannot cleave DNA anymore, retains its DNA binding capacity. Binding to the DNA strand will inhibit gene expression in a process called CRISPR interference [Larson (2013]. Because the dCas9 is essentially the same protein as the hCas9 from the CRISPR operon we expect that it will be able to use the same crRNA molecules as hCas9. However, expression of two different Cas9 variants has, to our knowledge, never been done before and unfortunately we did not have enough time to test the entirety of the system.

Another required change in our dCas9 concerns the PAM-recognition site. Kleinsteiver et al. described a set of four mutations (see table) of Cas9 that changed its PAM site from NGG into NGCG. In our project, we have combined this set of four mutations with the two dCas9 mutations resulting in dCas9VRER. To our knowledge, the combination of these sets of mutations has also not been attempted before.

A reporter construct is needed, which in its most simple form is a signal gene, whose expression can be modulated by CRISPR interference. This reporter essentially consists of a signal gene, a promotor and a predesigned spacer to which the dCas9 can bind. We developed a model which scored spacers based on likelihood of incorporation enabling us to choose the appropriate spacer.

A fourth sub-project that is not involved with the project directly is the Lactococcus 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 often do not work in L. lactis, promotors for example often need to be interchanged which also requires additional cloning steps and varying cloning protocols. Therefore, we have uploaded several protocols, obtained from our supervisor Patricia, and 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).