Difference between revisions of "Template:Team:Utrecht/MainBody"

 

In the OUTCASST system, our two DNA-sensing elements (dCas9 and dCpf1) are extracellularly fused to transmembrane proteins (see <a onclick="return change_page('outcasst', 1)" href="outcasst">OUTCASST system production</a>).

However, for proteins to reach the cell membrane, they have to pass through the Endoplasmic Reticulum and the Golgi apparatus <i class="ref" data-id="1">1</i>. The chemical environment inside these organelles differs greatly from that of the cytosol. This includes the possibility of glycosylation and disulfide bond formation to occur. For a functional OUTCASST, dCas9 and dCpf1 need to be able to pass through the secretion pathway, and still be able to bind gRNA complementary DNA afterwards.  

With these experiment, we aim to make HEK293t cells secrete Cas9 and Cpf1 into their growth medium. Subsequently, we will purify these secreted proteins and attempt to verify that they are still functional by performing an endonuclease assay. The proteins have maintained their DNA binding and cleaving capabilities if the proteins are still able to cut DNA strands in the correct place. If this is so, the catalytically inactive versions that form OUTCASST’s extracellular domains can be used in the final system, for we can then be sure that the secretory pathway does not disturb its DNA binding properties.

 

 

To be able to purify the secreted proteins from growth medium, a C-terminal His-tag with glycine linker (GGGHHHHHH) was added. Using this, it would be possible to purify the proteins using Ni-NTA affinity chromatography. Cas9 and Cpf1 containing the signal sequence and His-tag will henceforth be referred to as sCas9 and sCpf1, respectively.

 

<h2 class="subhead" id="subhead-3">Methods</h2>

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The amino acids sequences were also entered in the NetNGlyc 1.0 Server <i class="ref" data-id="4">4</i>, which predicts N-glycosylation sites based on the known motif (Asn-X-Ser/Thr-Ser/Thr), and also on the DiANNA 1.1 web server <i class="ref" data-id="5">5</i>, to highlight the cysteines and their likelihood to form disulfide bonds.

The predicted N-glycosylation sites were marked on crystal structures of both Cas9 and Cpf1 using PyMol <i class="ref" data-id="6">6</i> to get a clear image of where these sites would be and to clarify the likelihood that glycosylation would disturb protein functionality.

 

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DNA sequences coding for Cas9 and Cpf1 were modified to contain an N-terminal signal sequence (removing the first methionine from Cas9 and Cpf1) and a C-terminal His-tag. A kozak sequence was placed in front of the protein coding region, to help initiate the translation process in mammalian cells. This was then placed in a backbone plasmid (pCAGGS). These plasmids were subsequently amplified in <i>E. coli</i>. See the supplementary information for cloning procedures and plasmid sequences.

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HEK293t cells were cultured according to our cell culture protocol <a target=_BLANK href="https://static.igem.org/mediawiki/2017/2/2e/UU_Cell_culture_protocol.pdf" class="pdf pdf-inline"></a>.  

The cells were transfected with plasmids according to the Lipofectamine 2000 transfection protocol <a target=_BLANK href="https://static.igem.org/mediawiki/2017/1/17/UU_-_Lipofectamine_2000_transfection_protocol.pdf" class="pdf pdf-inline"></a>.

The first assay was performed according to protocol <a target=_BLANK href="https://static.igem.org/mediawiki/2017/1/12/Nuclease_activity_assay_-_assay_UU.pdf" class="pdf pdf-inline"></a>. An adjusted protocol was used for the second assay, due to low concentrations of purified sCas9 and sCpf1 <a target=_BLANK href="https://static.igem.org/mediawiki/2017/1/13/UU_-_Nuclease_activity_assay_-_assay2.pdf" class="pdf pdf-inline"></a>. See the supplementary information for cloning procedures and sequences.

<b>Secretion, glycosylation and disulfide bond prediction</b>

SignalP 4.1 predicted cleavage right after the signal sequence for both sCas9 and sCpf1. The results of the prediction can be found here <a target=_BLANK href="https://static.igem.org/mediawiki/2017/9/97/UU_-_SignalP_results.pdf" class="pdf pdf-inline"></a>.

Two cysteines were marked as having a high chance of forming disulfide bonds in Cas9, while  eight such cysteines were found in Cpf1. Details can be found in the supplementary information.

<span class="text-figure"><b>Figure 2. Western Blot using protein purified under denaturing conditions.</b> A band at the correct height is present in the lanes containing purified sCas9 and sCpf1 from the medium, indicating presence of sCas9 and sCpf1 in the medium.

Figure 2 shows the presence of sCas9 and sCpf1 in the medium, which indicated the proteins are secreted and that the experiment could be redone under native conditions but only small quantities of protein could be purified (~0,5 ng/uL in 0,5 mL for both sCas9 and sCpf1).

<span class="text-figure"><b>Figure 3. Western Blot using protein purified under native conditions.</b> The left image shows samples from cells transfected with a sCas9 containing plasmid, incubated with Anti-Cas9. The right image shows samples transfected with a sCpf1 containing plasmid, incubated with a Cpf1 antibody.The images suggest that sCas9 and sCpf1 are indeed secreted into the medium, though it cannot be excluded that part of the protein in the media is due to cell lysis.

After newly expressing sCas9 and sCpf1 in HEK293t cells, blots of the Cpf1 protein (figure 4) displayed the presence of secreted Cpf1 in both medium and cell lysate. In the medium, two bands are shown, one with a lower mass than Cpf1 is supposed to have. This lower band is, ostensibly, a degradation product. In the cell lysate, we see a second band as well, this time of higher mass. We suspect that this band corresponds to glycosylation product that accumulates in the cells and cannot be secreted. The results from the Wageningen_UR team confirm the presence of sCpf1 in both cell lysate and medium, as expected, again showing a lower mass band for the protein in the medium.

<span class="text-figure"><b>Figure 4. Secretion products of Cpf1.</b> The left image displays the blotting results of Utrecht’s team. The right image displays the blotting results of our Wageningen_UR collaborators. sCpf1 is shown on both blots, displaying two bands. Cell lysates of sCpf1 producing cells display sCpf1 as well.

<b>Endonuclease activity assay</b><br>

<center><img style="margin-top: 10px;" src="https://static.igem.org/mediawiki/2017/f/fc/UU_secretion_fig5.png"></center>

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<b>Figure 5.</b> Left - DNA gel electrophoresis of a linearized 800 base pair length plasmid. Concentrations of Cas9 and Cpf1 used in the assay were 0,05 uM and 0,15 uM, respectively. Right - DNA gel electrophoresis of the linearized 800bp plasmid. 2,5 nM Cas9 or Cpf1 protein was used and 10 nM gRNA.

<h2 class="subhead" id="subhead-5">Discussion</h2>

The goal of this experiment was to create secretable Cas9 and Cpf1 and test the functionality of these proteins. We have a clear indication that we achieved in obtaining secretion product, demonstrated by figures 2,3 and the collaboration results. Even if cell death has contributed to the protein concentrations in the media at all, it seems unlikely that it could account for a majority of the product. This, in addition to the lack of cell debris in the culturing plates, leads us to believe that the proteins can indeed be secreted.

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As of yet, we have not been able to demonstrate nuclease activity for either sCas9 or sCpf1. In our nuclease assay with the secreted proteins, the substrate was not cleaved. Even the control proteins did not show cleavage, suggesting that something went wrong with the assay. This lack of cleavage could be due to the low concentrations of protein purification product. In this assay, the incubation time was increased in comparison to standard endonuclease assay protocols to compensate for the low purification yields. Poor quality gRNA could also have caused the lack of cleavage. For future research, we suggest gRNA verification is performed before the assay is attempted. Based on our predictions for glycosylation, it seems likely that sCas9 and sCpf1 are still enzymatically active, even though some optimization might be required to gain satisfactory cleavage efficiency.

Versions of Cas9 and Cpf1 that can pass through the secretion pathway and maintain their DNA binding capabilities are essential for the OUTCASST system. The secretable sCas9 and sCpf1 proteins have merit on their own as well. A system where Cas9 and Cpf1 are made and secreted by mammalian cells could be useful for production of clinically usable endonucleases. Bacterial production may, at first glance, seem more straightforward but poses problems of clinical purity as bacterial endotoxins can easily contaminate the protein. Production of these proteins in mammalian cells could circumvent this problem.

 

<h2 class="subhead" id="subhead-6">References</h2>

 

<ol class="references">

<li data-title="Molecular Cell Biology." data-author="Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology." data-link="https://www.ncbi.nlm.nih.gov/books/NBK21471/" /> Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000. Section 17.3, Overview of the Secretory Pathway. <a target=_BLANK href="https://www.ncbi.nlm.nih.gov/books/NBK21471/" class="url_external"></a>

<li data-title="Modular Extracellular sensor architecture for engineering mammalian cell-based devices." data-author="Daringer, N. M., Dudek, R. M., Schwarz, K. A., & Leonard, J. N." data-link="https://doi.org/10.1021/sb400128g" /> Daringer, N. M., Dudek, R. M., Schwarz, K. A., & Leonard, J. N. (2014). Modular Extracellular sensor architecture for engineering mammalian cell-based devices. ACS Synthetic Biology, 3(12), 892–902. <a target=_BLANK href="https://doi.org/10.1021/sb400128g" class="url_external"></a>

<li data-title="SignalP 4.0: discriminating signal peptides from transmembrane regions." data-author="Petersen, T.N., Brunak, S., Von Heijne, G. & Nielsen, H." data-link="http://www.cbs.dtu.dk/services/SignalP/" /> Petersen, T.N., Brunak, S., Von Heijne, G. & Nielsen, H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nature Methods, 8:785-786, 2011. <a target=_BLANK href="http://www.cbs.dtu.dk/services/SignalP/" class="url_external"></a>

<li data-title="Prediction of N-glycosylation sites in human proteins." data-author="Gupta, R., Jung, E. and Brunak, S." data-link="http://www.cbs.dtu.dk/services/NetOGlyc/" /> Gupta, R., Jung, E. and Brunak, S. Prediction of N-glycosylation sites in human proteins. In preparation, 2004. <a target=_BLANK href="http://www.cbs.dtu.dk/services/NetOGlyc/" class="url_external"></a>

<li data-title="DiANNA 1.1: an extension of the DiANNA web server for ternary cysteine classification." data-author="Ferre, F. & Clote, P." data-link="http://clavius.bc.edu/~clotelab/DiANNA/" /> Ferre, F. & Clote, P. DiANNA 1.1: an extension of the DiANNA web server for ternary cysteine classification. Accepted for publication in Nucleic Acids Res. - Web Servers 2006 special issue. <a target=_BLANK href="http://clavius.bc.edu/~clotelab/DiANNA/" class="url_external"></a>

<li data-title="The PyMOL Molecular Graphics System." data-author="Schrödinger, LLC." data-link="https://pymol.org/2/" /> The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC. <a target=_BLANK href="https://pymol.org/2/" class="url_external"></a>

 

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<h2 class="subhead" id="subhead-7">Supplementary</h2>

 

 

5: "Discussion",

7: "Supplementary"