From 5ded313ec202e814897d2686b51faa74b815109d Mon Sep 17 00:00:00 2001
From: Philip Mundt <philip.mundt@uni-bielefeld.de>
Date: Sat, 28 Sep 2024 16:52:19 +0200
Subject: [PATCH] embedded nickase texts
---
src/contents/engineering.tsx | 133 +++++++++++++++++++++++++----------
1 file changed, 97 insertions(+), 36 deletions(-)
diff --git a/src/contents/engineering.tsx b/src/contents/engineering.tsx
index 760589bb..178397cc 100644
--- a/src/contents/engineering.tsx
+++ b/src/contents/engineering.tsx
@@ -4,12 +4,15 @@ import { LoremShort } from "../components/Loremipsum";
import { openElement } from "../utils/openElement";
import { H3, H4 } from "../components/Headings";
import { useTabNavigation } from "../utils/TabNavigation";
+import { TabScrollLink } from "../components/Link";
+import { useNavigation } from "../utils";
export function Engineering() {
useTabNavigation();
+ const {goToPagesAndOpenTab} = useNavigation();
return (
<>
<div className="row mt-4">
@@ -116,40 +119,40 @@ export function Engineering() {
<div className="box" >
<p id="pe1">
<H3 text="" id="text"/>
- <H4 text="Test" id="text"/>
- <p></p>
- <H4 text="Learn" id="text"/>
- <p></p>
<H4 text="Design" id="text"/>
<p></p>
<H4 text="Build" id="text"/>
<p></p>
+ <H4 text="Test" id="text"/>
+ <p></p>
+ <H4 text="Learn" id="text"/>
+ <p></p>
</p>
</div>
<div className="box" >
<p id="pe2">
<H3 text="" id="text"/>
- <H4 text="Test" id="text"/>
- <p></p>
- <H4 text="Learn" id="text"/>
- <p></p>
<H4 text="Design" id="text"/>
<p></p>
<H4 text="Build" id="text"/>
<p></p>
+ <H4 text="Test" id="text"/>
+ <p></p>
+ <H4 text="Learn" id="text"/>
+ <p></p>
</p>
</div>
<div className="box" >
<p id="pe3">
<H3 text="" id="text"/>
- <H4 text="Test" id="text"/>
- <p></p>
- <H4 text="Learn" id="text"/>
- <p></p>
<H4 text="Design" id="text"/>
<p></p>
<H4 text="Build" id="text"/>
<p></p>
+ <H4 text="Test" id="text"/>
+ <p></p>
+ <H4 text="Learn" id="text"/>
+ <p></p>
</p>
</div>
<br/>
@@ -173,50 +176,108 @@ export function Engineering() {
</div>
<div className="box" >
<p id="nik1">
- <h3>nik1</h3>
- <LoremShort></LoremShort>
- <p><LoremShort></LoremShort></p>
- <p><LoremShort></LoremShort></p>
+ <H3 text="SpuFz1 Zink Finger Mutation " id="text"/>
+ <H4 text="Design" id="text"/>
+ <p>In our quest to identify smaller endonucleases suitable for creating nickases, we focused on a newly characterized family of eukaryotic endonucleases known as Fanzor proteins, with SpuFz1 being a standout candidate due to its smaller size compared to Cas9. According to the paper "Fanzor is a Eukaryotic Programmable RNA-Guided Endonuclease"<TabScrollLink tab="tab-nikase" num="1" scrollId="desc-1"/>, SpuFz1 operates with two key domains: the RuvC domain, which cuts the non-target DNA strand, and the TNB domain (also called NUC domain), which helps facilitating the cleavage by pushing the target strand into the RuvC domain. We hypothesized that a zinc finger, which is part of the TNB domain and crucial for DNA coordination, could be a target for mutation to disrupt this process. Thus, our initial design strategy involved replacing the cysteine residues coordinating the zinc ion in the zinc finger with alanine to potentially impair its DNA-binding ability.</p>
+ <H4 text="Build" id="text"/>
+ <p>We used the protein visualization software ChimeraX to explore the SpuFz1 protein structure and identify the specific cysteine residues involved in zinc ion coordination. Based on this visualization, we designed mutant sequences by substituting these cysteines with alanine.</p>
+ <H4 text="Test" id="text"/>
+ <p>First, we discussed our approach with <a onClick={() => goToPagesAndOpenTab('hammer', '/human-practices')} >Kai Schülke</a>, a PhD student from the Hammer Group at Bielefeld University, which specializes in enzyme engineering. Although he could follow our logic, Kai mentioned that he was ultimately unable to assess the validity of our approach, as this specific class of enzymes falls outside his area of expertise. He recommended that, rather than testing a wide range of random mutations (due to the limited time of our project), we should focus on our specific mutant candidates by ordering the DNA sequences and cloning them into expression vectors.</p>
+ <p>Additionally, we simulated the potential effectiveness of our modified SpuFz1 nickase in a Prime Editing scenario, targeting the ΔF508 mutation in cystic fibrosis. During this simulation, we identified a challenge: the TAM sequence required for SpuFz1 binding was located relatively far from the target mutation site, which could reduce the effectiveness of the Prime Editor. However, we did not entirely rule out SpuFz1, considering that it might still be useful for other applications.</p>
+ <H4 text="Learn" id="text"/>
+ <p>From this iteration, we confirmed that targeted mutagenesis is the best approach for generating our mutant nickases. We also recognized the need for careful consideration of the PAM (or TAM) sequences associated with our chosen endonucleases. The realization that the TAM sequence of SpuFz1 might be too far from our target mutation suggests that SpuFz1 may not be the ideal candidate for this application, though it could still be an interesting candidate. Additionally, we learned the importance of expert consultation in refining our approach.</p>
</p>
</div>
<div className="box" >
<p id="nik2">
- <h3>nik2</h3>
- <LoremShort></LoremShort>
- <p><LoremShort></LoremShort></p>
- <p><LoremShort></LoremShort></p>
+ <H3 text="Fusion Protein from GtFz1 & SpuFz1" id="text"/>
+ <H4 text="Design" id="text"/>
+ <p>In our ongoing exploration of Fanzor proteins, we identified another potential candidate, GtFz1, which had a suitable TAM sequence for our target application of correcting the F508del mutation in cystic fibrosis. However, GtFz1 showed low cutting efficiency in the tests reported in the literature. To address this, we devised a strategy to combine the favorable TAM-binding region of GtFz1 with the higher cutting efficiency of SpuFz1. Specifically, we planned to engineer a fusion protein by replacing the TAM-binding domain of SpuFz1 with that of GtFz1. The idea was to create a new endonuclease with the optimal TAM sequence for our application and a robust DNA cutting ability.</p>
+ <p>Given that we were swapping entire domains rather than just single amino acids, we realized that the fusion protein might not retain the ideal TAM-binding efficiency or cutting efficiency of the original proteins. Our strategy was to create a fusion protein that could bind to the TAM site and perform DNA cutting to a certain extent, albeit weakly. We planned to use directed evolution techniques, such as Phage Assisted Continuous Evolution (PACE), to enhance these functionalities over time. This approach relies on having a starting point with some degree of the desired activity, which can then be incrementally improved through evolution.</p>
+ <H4 text="Build" id="text"/>
+ <p></p>
+ <H4 text="Test" id="text"/>
+ <p></p>
+ <H4 text="Learn" id="text"/>
+ <p></p>
</p>
</div>
<div className="box" >
<p id="nik3">
- <h3>nik3</h3>
- <LoremShort></LoremShort>
- <p><LoremShort></LoremShort></p>
- <p><LoremShort></LoremShort></p>
+ <H3 text="" id="text"/>
+ <H4 text="Design" id="text"/>
+ <p></p>
+ <H4 text="Build" id="text"/>
+ <p></p>
+ <H4 text="Test" id="text"/>
+ <p></p>
+ <H4 text="Learn" id="text"/>
+ <p></p>
</p>
</div>
<div className="box" >
<p id="nik4">
- <h3>nik4</h3>
- <LoremShort></LoremShort>
- <p><LoremShort></LoremShort></p>
- <p><LoremShort></LoremShort></p>
+ <H3 text="" id="text"/>
+ <H4 text="Design" id="text"/>
+ <p></p>
+ <H4 text="Build" id="text"/>
+ <p></p>
+ <H4 text="Test" id="text"/>
+ <p></p>
+ <H4 text="Learn" id="text"/>
+ <p></p>
</p>
</div>
<div className="box" >
<p id="nik5">
- <h3>nik5</h3>
- <LoremShort></LoremShort>
- <p><LoremShort></LoremShort></p>
- <p><LoremShort></LoremShort></p>
+ <H3 text="" id="text"/>
+ <H4 text="Design" id="text"/>
+ <p></p>
+ <H4 text="Build" id="text"/>
+ <p></p>
+ <H4 text="Test" id="text"/>
+ <p></p>
+ <H4 text="Learn" id="text"/>
+ <p></p>
</p>
</div>
<div className="box" >
<p id="nik6">
- <h3>nik6</h3>
- <LoremShort></LoremShort>
- <p><LoremShort></LoremShort></p>
- <p><LoremShort></LoremShort></p>
+ <H3 text="" id="text"/>
+ <H4 text="Design" id="text"/>
+ <p></p>
+ <H4 text="Build" id="text"/>
+ <p></p>
+ <H4 text="Test" id="text"/>
+ <p></p>
+ <H4 text="Learn" id="text"/>
+ <p></p>
+ </p>
+ </div>
+ <div className="box" >
+ <p id="nik6">
+ <H3 text="References" id="text"/>
+ <ol>
+ {/*<!-- Citation num 1--> */}
+ <li typeof="schema:ScolarlyArticle" role="doc-biblioentry" property="schema:citation" id="desc-1">
+ <span property="schema:author" typeof="schema:Person">
+ <span property="schema:Name"> Saito, M.</span>
+ <span property="schema:Name"> Xu, P.</span>
+ <span property="schema:Name"> Faure, G.</span>
+ <span property="schema:Name"> Maguire, S.</span>
+ <span property="schema:Name"> Kannan, S.</span>
+ <span property="schema:Name"> Altae-Tran, H.</span>
+ <span property="schema:Name"> Vo, S.</span>
+ <span property="schema:Name"> et al.</span>
+ </span>
+ <span property="schema:name"> Fanzor is a eukaryotic programmable RNA-guided endonuclease</span>.
+ <i property="schema:publisher" typeof="schema:Organization"> Nature</i>
+ <b property="issueNumber" typeof="PublicationIssue"> 620</b>
+ , <span property="schema:pageBegin"> 660</span>-<span property="schema:pageEnd">668</span>
+ (<time property="schema:datePublished" datatype="xsd:gYear" dateTime=" 2023">2023</time>).
+ <a className="doi" href="https://doi.org/10.1038/s41586-023-06356-2"> doi: 10.1038/s41586-023-06356-2</a>
+ </li>
+ </ol>
</p>
</div>
<br/>
--
GitLab