Difference between revisions of "Part:BBa K1062004"

Csy4 (Csy6f), a member of CRISPR family.

This year, we create a brand new family called Csy4 family on the basis of an existing part, Csy4 BBa_K1062004. We redesign four Csy4 mutants by point mutation. The members in Csy4 family have different capabilities of cleavage and recognition. As an important role in project, we tested them by several ways. The Csy4 family works well as expectation. Csy4 family is an improvement based on existing part and is proved work well in our system.

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 mutants

We did three kinds of experiments to help us confirm the function of the Csy4 family. The aim is to get some new Csy4 mutants with different capabilities. Superfolder green fluorescent protein (sfGFP) is target gene for test experiments. Our expectation is that the fluorescence intensities of sfGFP change upon various activity of Csy4 mutants. It means we have improved four new parts which present various expression of target genes.

Prediction

Before the experiments, model proved our ideas. The predication shows the possibilities of different expression levels by different Csy4 mutants. It is not difficult to predict that the cleavage rate has an influence in the expression of sfGFP.

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

We designed three kinds experiments to test the capabilities of five Csy4 mutants by putting them into miniToe system. So the recombination strains for test both have same pReporter but different Csy4 mutants plasmids in the following. The recombination strains to test the functions of Csy4 are strain-Csy4 (pCsy4&pReporter), strain-Csy4-Q104A (pCsy4-Q104A&pReporter), strain-Csy4-Y176F (pCsy4-Y176F&pReporter), strain-Csy4-F155A (pCsy4-F155A&pReporter), strain-Csy4-H29A (pCsy4-H29A&pReporter). At the same time, we have a control strain named strain-miniToe-only which only has pReporter.

The qualitative experiments by fluorescent microscope

First, we have tested five different groups by Fluorescent Stereo Microscope Leica M165 FC. The sfGFP accumulated during the cultivation period so the fluorescence can be observed by microscope after 8 hours. Because the five Csy4 mutants have different capabilities of cleavage, the distinguishing intensities of fluorescent can be seen by naked eyes. The five test strains have same miniToe part but different Csy4 mutant genes. From top to bottom in Fig.5, there are fluorescence images by fluorescent microscope which indicate strain-Csy4, strain-Csy4-Q104A, strain-Csy4-Y176F, strain-Csy4-F155A and strain-Csy4-H29A in sequence. The visible distinctions have shown 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. The Csy4-WT has the strongest capability of cleavage when 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 strain-Csy4.

Fig.5-2 The expression of sfGFP by strain-Csy4-Q104A.

Fig.5-3 The expression of sfGFP by strain-Csy4-Y176F.

Fig.5-4 The expression of sfGFP by strain-Csy4-F155A.

Fig.5-5 The expression of sfGFP by strain-Csy4-H29A.

From top to bottom, the images shows the expression of sfGFP by strain-Csy4, strain-Csy4-Q104A, strain-Csy4-Y176F, strain-Csy4-F155A and strain-Csy4-H29A in sequence. The plotting scale is on the right corner of images. The images on the left shows E. coli without fluorescence excitation. The images on the right represent situation when fluorescence excitation.

The result by flow cytometer

The qualitative experiment is not enough to analyze the Csy4 mutants. So we tested miniToe family system by flow cytometer. The expression of sfGFP by strain-Csy4, strain-Csy4-Q104A, strain-Csy4-Y176F, strain-Csy4-F155A and strain-Csy4-H29A is showed in Fig.6. We find that 5 groups' fluorescence intensities have an obvious order from Csy4-WT to Csy4-H29A, which means the capabilities decrease one by one. Their order goes from strong to weak is Csy4-WT, Csy4-Q104A, Csy4-Y176F,Csy4-F155A and Csy4-H29A.

Fig.6 The fluorescence intensities of sfGFP about Csy4 mutants by flow cytometer. Histograms show distribution of fluorescence in samples with strain-Csy4 (Black), strain-Csy4-Q104A (Orange), strain-Csy4-Y176F (Red), strain-Csy4-F155A (Blue), strain-Csy4-H29A (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 before, we also need to know more information about the Csy4 mutants in entire cultivation period. Even though we known that our Csy4 mutants have differentiated expression level in ten-hour-culture, the expression of whole cultivation period is also a reference for us to know if our system can work as expectations.

So we tested five test stains individually (strain-Csy4, strain-Csy4-Q104A, strain-Csy4-Y176F, strain-Csy4-F155A and strain-Csy4-H29A) by microplate reader every two hours. The green lines in all the images represents strain-miniToe-only group keep stable. It means the miniToe structure fold well and lock the process of translation without Csy4. And the five test groups show different characteristics. In Fig.8-A, the group strain-Csy4 shows the same result with the first system. The switch turns off without IPTG (as the blue line shows). And the expression level is the highest among all the test groups which indicates the Csy4-WT has strongest capabilities (Fig.8-F). In the Fig.8-B, the tendency of fluorescence intensities by Csy4-Q104A is similar with Csy4-WT. And the expression level is lower than Csy4-WT. The Csy4-Y176F’s capabilities ranks the third. What is special is Csy4-H29A. 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 prediction by model match the result perfectly in Fig.9.

Fig.8 The fluorescence intensities of sfGFP by microplate reader. A. strain-Csy4. B. strain-Csy4-Q104A. C. strain-Csy4-Y176F. D. strain-Csy4-F155A. E. strain-Csy4-H29A. 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. The results are listed in the order: Csy4-WT>Csy4-Q104A>Csy4-Y176F>Csy4-F155A>Csy4-H29A.

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

By all the experiments mentioned before, we proved that Csy4 mutants work as expectations successfully. The results are listed in the order: Csy4-WT>Csy4-Q104A>Csy4-Y176F>Csy4-F155A>Csy4-H29A. And the original sequences of Csy4 part has been submitted by other iGEM teams before, so this year we improved their work by enlarging Csy4 to a Csy4 family.

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.