Difference between revisions of "Part:BBa K1062004"

Csy4 (Csy6f), a member of CRISPR family.

===Csy4 (Csy6f), a member of CRISPR family.=== <p> The endoribonuclease Csy4 from CRISPR family is the main role of miniToe system. Csy4 (Cas6f) is a 21.4 kDa protein which recognizes and cleaves a specific 22nt RNA hairpin. In type I and type III CRISPR systems, the specific Cas6 endoribonuclease splits the pre-crRNAs in a sequence-specific way to generate 60-nucleotide (nt) crRNA products in which segments of the repeat sequence flank the spacer (to target "foreign" nucleic acid sequence) [1]. Inactivation of the Cas proteins leads to a total loss of the immune mechanism function.

The Csy4 protein consists of an N-terminal ferredoxin-like domain and a C-terminal domain. This C-terminal domain is responsible for pre-crRNA recognition and binding. The pre-crRNA target site adopts a stem-loop structure (the specific 22nt RNA hairpin) with five base pairs in A-form helical stem capped by GUAUA loop containing a sheared G11-A15 base pair and a bulged nucleotide U14. In the binding structure of Csy4-RNA complex, the RNA stem-loop straddles the β-hairpin formed by strands β6-7 of Csy4[2]. And once the Csy4/RNA complex formed, the structure will stay stable and hard to separate.

[1].Przybilski R, Richter C, Gristwood T, et al. Csy4 is responsible for CRISPR RNA processing in Pectobacterium atrosepticum.[J]. Rna Biology, 2011, 8(3):517-528.

[2].Haurwitz R E, Jinek M, Wiedenheft B, et al. Sequence- and structure-specific RNA processing by a CRISPR endonuclease[J]. Science, 2010, 329(5997):1355-1358.

Fig.1 The Csy4/Hairpin complex.

Background of 2018 OUC-China' project

This year, we designed and achieved a gene regulatory toolbox based on CRISPR RNA endonucleases Csy4 for post-transcriptional regulation. A rational designed modular RNA fragment named miniToe was utilized for precise and efficient translational regulation. The miniToe module was constructed through inserting a 22 nt Csy4 recognition site between RBS and a cis-repressive RNA element, which is able to mask the RBS region and inhibit translation initiation. By up-regulating the level of Csy4 in cell as input, the miniToe module will be cleaved and releases an exposed RBS for output translation. As our innovation, we further designed four Csy4 mutants and five mutated miniToe module in a predictable way by modeling, which aims at enriching our toolkit for diverse regulation ranges on target genes. The whole toolbox includes ten combinations of different Csy4 mutants and miniToe modules, which is called miniToe family.

As a key role in miniToe system, some key sites in the Csy4 are really crucial for keeping the stable of structure and maintain the functions of recognition and cleavage. Mutatios on those sites may result in serious influence on our system. By point mutation, we hope to get a library of mutants, which could provide several Csy4 mutant candidates with recognition and cleavage rates shows as a "ladder".

Here a model helps to design the mutants of Csy4 and hairpin. According to the principles of design we mentioned above, four key problems is important in the miniToe model design:

1.Does the Csy4 dock correctly with the miniToe structure?
2.How about the binding ability between the Csy4 and miniToe structure?
3.How about the ability of cleavage between the Csy4 and miniToe structure?
4. Does cis-repressive release from the RBS?

For the Csy4 mutants, the molecular dynamics method was used as our tools. Based on Ji?í ?poner's work [1], we chose four significant symbols of mutants: the interaction matrix; the binding free energy, the distance of Ser151(OG)-G20(N2') and the RMSD of the cleaved-product complex. By comparing the difference between various Csy4 mutants with wild-type Csy4 by above four significant symbols, we finally design four Csy4 mutants: Csy4-Q104A, Csy4-Y176F, Csy4-F155A, and Csy4-H29A based on model prediction.

Fig.2 Four key sites of wild type Csy4.

Proof of functions about Csy4 family

We have done three kinds of experiments to help us confirm the function of the Csy4 family. Our aim is to get some new Csy4 mutants with different cleavage capacity, so we specifically tested this aspect of them. For testing our system, we use the superfold green fluorescent protein (sfGFP) as our target gene. Our expectation is that the fluorescence intensities of sfGFP can vary upon the rates of Csy4s’ cleavage. That means we have improved four new parts which present various expression of target genes.

Prediction

Before the experiments, we have proved our ideas by model. The predication below shows the possibilities of different expression levels by different Csy4 mutants. So the model help us to get more information for our improvement deeply this year!

Fig.4 The predication: The fluorescence intensities by different Csy4 mutants along with time.

The qualitative experiments by fluorescent microscope

First, we have tested five different Csy4s by Fluorescent Stereo Microscope Leica M165 FC. We have cultured them in the solid medium in plates until the bacterial colonies can be observed by naked eyes. At that time, the sfGFP have been accumulated so we can see the fluorescence by microscope. As we can see in Fig.5, we have cultured the five different strains for same time which both have the same miniToe circuit but have totally different Csy4 mutants. From top to bottom in Fig.5, there are fluorescence images by fluorescent microscope which indicate Csy4-WT, Csy4-Q104A, Csy4-Y176F, Csy4-F155A and Csy4-H29A in sequence. We can observe visible distinctions in these images. The fluorescence intensities decrease one by one from top to bottom which means the Csy4s’ capabilities of cleavage decrease one by one. So the images indicate the Csy4-WT has the strongest capability of cleavage, while the Csy4-H29A is a kind of dead-Csy4 (dCsy4) which is hardly to find the fluorescence by microscope. The qualitative experiment is a basis of further experiments.

Fig.5-1 The expression of sfGFP by Csy4-WT&miniToe-WT.

Fig.5-2 The expression of sfGFP by Csy4-Q104A&miniToe-WT.

Fig.5-3 The expression of sfGFP by Csy4-Y176F&miniToe-WT.

Fig.5-4 The expression of sfGFP by Csy4-F155A&miniToe-WT.

Fig.5-5 The expression of sfGFP by Csy4-H29A&miniToe-WT.

The result by flow cytometer

The qualitative experiment is not enough to analyze Csy4s. So we test our system by flow cytometer after we cultured them for ten hours in M9 medium. The expression of sfGFP in five groups are showed in Fig.6, and they are Csy4-WT&miniToe-WT, Csy4-Q104A&miniToe-WT, Csy4-Y176F&miniToe-WT, Csy4-F155A&miniToe-WT and Csy4-H29A&miniToe-WT. We find that 5 groups’ fluorescence intensities have an obvious order from Csy4-WT to Csy4-H29A, which means the capabilities of cleavage decrease one by one. Their order goes from strong to weak is Csy4-WT, Csy4-Q104A, Csy4-Y176F, Csy4-F155A and Csy4-H29A. As the Fig.6 shown, the relative expression level can be measured by flow cytometer at the same time.

Fig.6 Fluorescence intensity of sfGFP corresponding to each Csy4. Histograms show distribution of fluorescence in samples with Csy4-WT&miniToe-WT (Black), Csy4-Q104A&miniToe-WT (Orange), Csy4-Y176F&miniToe-WT (Red), Csy4-F155A&miniToe-WT (Blue), Csy4-H29A&miniToe-WT (Green). Crosscolumn number shows fold increase of sfGFP fluorescence.

Fig.7 The Gate Mean of flow cytometer. Histograms show the relative expression of sfGFP. The five test groups present different fluorescence intensities from high to low, which prove that they have different capabilities of cleavage.

The result by microplate reader

Besides all the works we have done before, we also need to know more information about the Csy4s we design. Even though we have known that our Csy4 mutants have differentiated expression level after ten-hour-culture, the expression of whole cultivation period is also a reference for us to know if our system can work as expectation.

So we tested five Csy4s individually by microplate reader. We have tested them every two hours. The green lines in all the images represent the control group, “miniToe only” group and the green lines keep stable which means the miniToe structure can close the expression of downstream genes. And the test groups show different characteristics. As we can see in Fig.8-A, the Csy4-WT shows the same result with the first system. The switch turn off when the system without isopropyl-β-d-thiogalactoside (IPTG) (as the blue line shows). And the expression level is the highest among all the test groups which indicates the highest enzyme activity by Csy4-WT (Fig.8-F). In the Fig.8-B, the tendency of increase of fluorescence intensities by Csy4-Q104A is almost same with Csy4-WT. And the expression level is lower than Csy4-WT. So the Csy4-Y176F is. What is special is Csy4-H29A. We have mentioned Csy4-H29A before. The active site of Csy4 contains an essential histidine residue (H29) that functions as a general base during RNA strand scission. Mutation of H29 to alanine inactivates Csy4 without affecting substrate binding affinity or specificity. So Csy4-H29A is a dead-Csy4 which has high binding affinity but has lowest capabilities of cleavage as we can see in Fig.8-E. In summary, we put all the test groups together in Fig.8-F, the picture shows our prediction by model matchs the result perfectly in Fig.9.

Fig.8 The fluorescence intensities of sfGFP by microplate reader. A. Csy4-WT&miniToe-WT. B. Csy4-Q104A&miniToe-WT. C. Csy4-Y176F&miniToe-WT. D. Csy4-F155A&miniToe-WT. E. Csy4-H29A&miniToe-WT. A-E. The blue line is test group with IPTG. The yellow line is test group without IPTG. The green line is a control group which only has miniToe structure without Csy4s. F. The summary of different test groups which indicates the capabilities of Csy4 mutants.

Fig.9 The comparison about model and result by microplate reader.

In summary

This year, we used point mutations to redesign four mutants on the basis of Csy4(BBa_K1062004) which are Csy4-Q104A(BBa_K2615004), Csy4-Y176F(BBa_K2615005), Csy4-F155A(BBa_K2615006) and Csy4-H29A(BBa_K2615007). The capabilities of cleavage and recognition are different for each Csy4 mutants, and we name them the Csy4 family. The combination of the Csy4 family members and the miniToe family members constitute a post-transcriptional regulatory toolkit for achieving different expression levels of target genes.

Csy4-WT, the wild type, is a member of the CRISPR family, and also the core member of our project. Csy4-WT can specifically recognize and cleave a 22nt hairpin structure, known as the miniToe-WT. We confirmed that Csy4-WT is the strongest of the Csy4 family through the analysis of the results of our fluorescence microscopy, flow cytometry and microplate reader experiments. And the strength of the remaining members of the Csy4 family shows a staircase pattern.

Csy4-Q104A, which is second only to Csy4-WT in strength in the Csy4 family, coming from point mutation, and we change the CAG(encoding Gln) to GCG(encoding Ala) on the 104th site based on Csy4-WT. It can also recognize and cleave the 22nt miniToe, regulating the expression of downstream genes. When we conducted experiments with the miniToe-WT combination and used sfGFP as the downstream target gene, we could see the experimental results that the sfGFP expression level of Csy4-Q104A was about half that of Csy4-WT.

Csy4-Y176F, the third-strongest in the Csy4 family. It is designed in the same way as Csy4-Q104A, but with the 176th site changed from TAC(encoding Tyr) to TTT(encoding Phe). It can be seen from the experimental results that the expression of downstream genes regulated by Csy4-Y176F is correlated with the stepwise decline of Csy4-WT and Csy4-Q104A.

Csy4-F155A, strength is the fourth in the Csy4 family. At point mutation, we changed its 155th site from TTC(encoding Phe) to GCG(encoding Ara). It has a weaker cleavage and recognition capability.

Csy4-H29A, the most special one of our Csy4 family, whose 29th site is changed from CAC(encoding His ) to GCG(encoding Ara). Csy4-H29A has a high binding affinity but has the lowest capacity of cleavage, so we call it dead-Csy4. There is no doubt that its downstream gene expression is the lowest in the family.