Research

Project Description

Our IMPACT-system consists of cells that are capable of detecting phage infections. In this section, we will elaborate how we want to construct this system. On a biological level, our system can be divided into three parts. First, we need to detect the phages for which we will use a slightly adapted version of the S. pyogenes CRISPR-Cas system. Instead of using the normal CRISPR system we will resort to using a hyperactive Cas9 (hCas9). Heler et al. described a single amino acid mutation that turns Cas9 into hCas9, which resulted in an ~100 fold increase of spacer uptake rate [Heler (2017)].

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 (link: Larson et al.(2013). CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nature Protocols, [online] 8(11), pp.2180-2196.). 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).