Team:Groningen/Description

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Research

Project Description

Our IMPACT-system contains cells that are capable of detecting phage infections. In this section we will explain how we want to construct these cells. On a biological level our system can be divided into three parts. First we need to detect the phages for this we will use a slightly adaptive 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)].

Next the signal, which is the spacer at this moment, needs to be processed. This will be done by a second Cas9 variant, namely dCas9. The catalytically dead Cas9, which is a result of two mutations (table), cannot cleave DNA anymore, but it can interfere with gene expression in a process called CRISPR interference. Because the dCas9 is essentially exactly the same protein as the hCas9 from the CRISPR operon we expect that it can use the same CrRNA molecules as hCas9. However expressing two different Cas9 variants has, to our knowledge, never been done before and unfortunately we did not have enough time to test it.

Table: Cas9 variants and their corresponindg mutations

name	mutations	description
hCas9	I473F	Hyperactive Cas9
dCas9	D10A, H840A	Catalytically inactive variant of Cas9
Cas9[VRER]	D1135Y, G1218R, R1135E, T1337R	Cas9 9 with changed PAM specificity

Another thing that was changed in our dCas9 is the PAM-recognition site. Kleinsteiver et al. described a set of four mutations (see table) in the end of Cas9 that changed its PAM preference 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 is also new, but we did show that it work hopefully ☺.

Finally a reporter part is needed, which in its most simple form is a coloured gene, whose expression can be influenced by CRISPR interference. Something about target array plus introduce model.

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.

References

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).

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