diff --git a/src/components/makeSources.tsx b/src/components/makeSources.tsx
index bc4cb7ddb31294d7a41fd59270dd64984f423a86..6f2cf70cefbe511fd9993e8ee1658fdfce67ac65 100644
--- a/src/components/makeSources.tsx
+++ b/src/components/makeSources.tsx
@@ -15,7 +15,8 @@ interface BibEntry {
interface BibtexParserProps {
bibtexSources: string[]; // Accept an array of BibTeX strings
- special?: string
+ special?: string,
+ start?: number
}
@@ -54,7 +55,7 @@ function formatPages(pages: string | undefined): JSX.Element | null {
}
-export const BibtexParser: React.FC<BibtexParserProps> = ({ bibtexSources , special}) => {
+export const BibtexParser: React.FC<BibtexParserProps> = ({ bibtexSources , special, start}) => {
const [parsedEntries, setParsedEntries] = useState<BibEntry[]>([]);
// Parse BibTeX on component mount or when sources change
@@ -138,7 +139,10 @@ export const BibtexParser: React.FC<BibtexParserProps> = ({ bibtexSources , spec
// console.log(`Rendering citation for entry #${index + 1}: `, entry);
// Use the index as citation number
- const citationNumber = index + 1;
+ let citationNumber = index +1;
+ if(start){
+ citationNumber += start -1;
+ }
const entryType = entry.entryType.toLowerCase(); // Convert to lowercase for consistent comparison
const entryTags = entry.entryTags; // Adjust based on your data structure
// console.log("Entry Tags: ", entryTags);
@@ -240,13 +244,16 @@ export const BibtexParser: React.FC<BibtexParserProps> = ({ bibtexSources , spec
return <li key={index}>Unknown entry type: {entryType}</li>;
}
};
-
+ let startnumber = 1;
+ if(start) {
+ startnumber = start;
+ }
return (
<div>
{parsedEntries.length === 0 ? (
<p>No citations available.</p>
) : (
- <ol>
+ <ol start={startnumber}>
{parsedEntries.map((entry, index) => renderCitation(entry, index))}
</ol>
)}
diff --git a/src/contents/engineering.tsx b/src/contents/engineering.tsx
index 3671c2a928aebfa9a24e01df509610748c949428..30d196aa06772815eea0170f676a3747137c6654 100644
--- a/src/contents/engineering.tsx
+++ b/src/contents/engineering.tsx
@@ -248,7 +248,7 @@ export function Engineering() {
<div className="eng-box box" >
<H2 id="transfection-header" text="Optimization of Transfection"></H2>
<p>
- To test prime editors, a reliable model system is required. HEK293 cells are a human derived cell line and widely used in a variety of fields in biology<TabScrollLink tab="tab-transfection" num="1" scrollId="desc-1"/>. Apart from easy handling and comparatively easy transfection, they have, as we found out in our exchange with <a onClick={() => goToPagesAndOpenTab('mattijsinv', '/human-practices')}>Mattijs Bulcaen</a>, one advantage over other models: They are naturally impaired in DNA repair mechanisms and therefore easier to edit. To properly compare editing efficiencies, a high transfection efficiency is of utmost importance. This engineering cycle focuses on our work in simulating prime editing using the PEAR reporter system<TabScrollLink tab="tab-transfection" num="2" scrollId="desc-2"/> and optimizing transfection protocols.
+ To test prime editors, a reliable model system is required. HEK293 cells are a human derived cell line and widely used in a variety of fields in biology<TabScrollLink tab="transfection" num="3" scrollId="desc-3"/>. Apart from easy handling and comparatively easy transfection, they have, as we found out in our exchange with <a onClick={() => goToPagesAndOpenTab('mattijsinv', '/human-practices')}>Mattijs Bulcaen</a>, one advantage over other models: They are naturally impaired in DNA repair mechanisms and therefore easier to edit. To properly compare editing efficiencies, a high transfection efficiency is of utmost importance. This engineering cycle focuses on our work in simulating prime editing using the PEAR reporter system<TabScrollLink tab="transfection" num="4" scrollId="desc-4"/> and optimizing transfection protocols.
</p>
</div>
<div className="box" >
@@ -263,7 +263,7 @@ export function Engineering() {
</p>
<H4 text="Build" id="text"/>
<p>
- Anzalone et al. 2019<TabScrollLink tab="tab-transfection" num="3" scrollId="desc-3"/> describe a transfection of prime-editing complexes with Lipofectamine 2000.
+ Anzalone et al. 2019<TabScrollLink tab="transfection" num="5" scrollId="desc-5"/> describe a transfection of prime-editing complexes with Lipofectamine 2000.
</p>
<H4 text="Test" id="text"/>
<p>
@@ -364,9 +364,12 @@ export function Engineering() {
<section id="PE Systems sec" >
<div className="eng-box box" >
<H2 id="pe-systems-header" text="Prime Editing Systems"></H2>
- <p>Different versions of the original prime editing system have been developed since its initial introduction. Deciding on what system to use for the application in therapeutic human gene editing, especially concerning the correction of F508del, was the goal of this engineering cycle.</p>
+ <p>Different versions of the original prime editing system have been developed since its initial introduction.
+ Deciding on what system to use for the application in therapeutic human gene editing, especially concerning the correction of
+ F508del, was the goal of this engineering cycle.</p>
<p>
- Since we aim to develop a therapy delivered to the human body, we wanted to obtain high editing efficiency while risking as little off-targets as possible and also reducing the size for improved packability.
+ Since we aim to develop a therapy delivered to the human body, we wanted to obtain high editing efficiency while risking as
+ little off-targets as possible and also reducing the size for improved packability.
</p>
<InfoBox title="Existing Prime Editing Systems" id="current-pe-systems">
<details>
@@ -376,10 +379,10 @@ export function Engineering() {
<div className='row align-items-center'>
<div className='col'>
<p>
- <b>PE1</b><TabScrollLink tab="tab-pe-systems" num="1" scrollId="desc-1"/>, the first version of the Prime Editor features a Cas9(H840A), a Streptococcus pyogenes Cas9 (SpCas9, hereafter just referred to as Cas9) mutant that only cuts the non-target strand of the DNA template<TabScrollLink tab="tab-pe-systems" num="2" scrollId="desc-2"/>, and a wildtype reverse transcriptase from the Moloney Murine Leukaemia Virus (M-MLV RT) connected by a serine and glycine rich flexible linker.
+ <b>PE1</b><TabScrollLink tab="tab-pe-systems" num="6" scrollId="desc-6"/>{/* ehem 1 */}, the first version of the Prime Editor features a Cas9(H840A), a Streptococcus pyogenes Cas9 (SpCas9, hereafter just referred to as Cas9) mutant that only cuts the non-target strand of the DNA template<TabScrollLink tab="tab-pe-systems" num="7" scrollId="desc-7"/>{/* ehem 2 */}, and a wildtype reverse transcriptase from the Moloney Murine Leukaemia Virus (M-MLV RT) connected by a serine and glycine rich flexible linker.
</p>
<p>
- <b>PE2</b><TabScrollLink tab="tab-pe-systems" num="1" scrollId="desc-1"/> improves on this concept by incorporating an improved RT with five mutations improving affinity to the template RNA, enzyme processivity and thermostability (D200N/L603W/T330P/T306K/W313F). This version of the prime editor showed varying improvement of editing efficiency over all tested loci and edits with no apparent downsides. Building on the PE2 system, a smaller version of the M-MLV RT was introduced by Gao et al. (2022)<TabScrollLink tab="tab-pe-systems" num="3" scrollId="desc-3"/>. The RT was truncated by 621 bp through deletion of the RNaseH domain, which originally degrades the RNA template, but is not needed for prime editing. The codon optimized version of this truncated RT prime editor (in literature usually called PE2∆RNaseH) was named <b>PE<sup>CO</sup>-Mini</b> in the paper and will be addressed as such here.
+ <b>PE2</b><TabScrollLink tab="tab-pe-systems" num="6" scrollId="desc-6"/>{/* ehem 1 */} improves on this concept by incorporating an improved RT with five mutations improving affinity to the template RNA, enzyme processivity and thermostability (D200N/L603W/T330P/T306K/W313F). This version of the prime editor showed varying improvement of editing efficiency over all tested loci and edits with no apparent downsides. Building on the PE2 system, a smaller version of the M-MLV RT was introduced by Gao et al. (2022)<TabScrollLink tab="tab-pe-systems" num="8" scrollId="desc-8"/>{/* ehem3 */}. The RT was truncated by 621 bp through deletion of the RNaseH domain, which originally degrades the RNA template, but is not needed for prime editing. The codon optimized version of this truncated RT prime editor (in literature usually called PE2∆RNaseH) was named <b>PE<sup>CO</sup>-Mini</b> in the paper and will be addressed as such here.
</p>
</div>
<div className='col-4'>
@@ -392,7 +395,7 @@ export function Engineering() {
<div className='row align-items-center'>
<div className='col'>
<p>
- The <b>PE3</b><TabScrollLink tab="tab-pe-systems" num="1" scrollId="desc-1"/> system, described in the same paper as PE1 and PE2, introduces the use of a second single guide RNA besides the pegRNA which leads to a nick in the strand opposite to the edited strand. This is supposed to improve integration of edits by directing cellular DNA repair systems to use the edited strand as a template for resolving base mismatches. Nicks positioned 3‘ of the edit about 40–90 base pairs from the pegRNA-induced nick were able to further increase editing efficiencies about threefold when compared to PE2, but with a higher range of on-target indels , meaning random Insertions and/or Deletions that appear after faulty repair of double strand breaks in the DNA. PE3b, where the protospacer for the nicking sgRNA lies within the edited regions, decreased the indel rate greatly compared to PE3.
+ The <b>PE3</b><TabScrollLink tab="tab-pe-systems" num="6" scrollId="desc-6"/>{/* ehem 1 */} system, described in the same paper as PE1 and PE2, introduces the use of a second single guide RNA besides the pegRNA which leads to a nick in the strand opposite to the edited strand. This is supposed to improve integration of edits by directing cellular DNA repair systems to use the edited strand as a template for resolving base mismatches. Nicks positioned 3‘ of the edit about 40–90 base pairs from the pegRNA-induced nick were able to further increase editing efficiencies about threefold when compared to PE2, but with a higher range of on-target indels , meaning random Insertions and/or Deletions that appear after faulty repair of double strand breaks in the DNA. PE3b, where the protospacer for the nicking sgRNA lies within the edited regions, decreased the indel rate greatly compared to PE3.
</p>
</div>
<div className='col-4'>
@@ -402,10 +405,10 @@ export function Engineering() {
<div className='row align-items-center'>
<div className='col'>
<p>
- <b>PE4</b> and <b>PE5</b><TabScrollLink tab="tab-pe-systems" num="4" scrollId="desc-4"/> expand the PE2 and PE3 systems, respectively, by co-expressing a dominant negative MLH1 protein (MLH1(Δ754–756), hereafter referred to as MLH1dn). The MLH1 protein plays a crucial role in the mismatch repair (MMR) mechanism of the human cell<TabScrollLink tab="tab-pe-systems" num="5" scrollId="desc-5"/> by recruiting other repair proteins and facilitating catalytic function. The mutant still recruits other factors but is impaired in its endonuclease function, disrupting function of the entire repair mechanism. This leads to an average 7.7-fold and 2.0-fold increase in editing efficiency, respectively, compared to PE2 and PE3. This is possibly due to slower repair of mismatches and thus more time for the proteins encoded by LIG1 and FEN1 genes to excise the non-edited 5’ flap and ligate the nick in the edited strand. Additionally, MLH1dn co-expression slightly reduced on-target indels as well as unintended editing outcomes in PE3 systems and did not lead to higher off-target indel rates or overall mutation rates.
+ <b>PE4</b> and <b>PE5</b><TabScrollLink tab="tab-pe-systems" num="8" scrollId="desc-8"/> {/* ehem 4 */}expand the PE2 and PE3 systems, respectively, by co-expressing a dominant negative MLH1 protein (MLH1(Δ754–756), hereafter referred to as MLH1dn). The MLH1 protein plays a crucial role in the mismatch repair (MMR) mechanism of the human cell<TabScrollLink tab="tab-pe-systems" num="9" scrollId="desc-9"/>{/* ehem 5 */} by recruiting other repair proteins and facilitating catalytic function. The mutant still recruits other factors but is impaired in its endonuclease function, disrupting function of the entire repair mechanism. This leads to an average 7.7-fold and 2.0-fold increase in editing efficiency, respectively, compared to PE2 and PE3. This is possibly due to slower repair of mismatches and thus more time for the proteins encoded by LIG1 and FEN1 genes to excise the non-edited 5’ flap and ligate the nick in the edited strand. Additionally, MLH1dn co-expression slightly reduced on-target indels as well as unintended editing outcomes in PE3 systems and did not lead to higher off-target indel rates or overall mutation rates.
</p>
<p>
- With <b>PEmax</b><TabScrollLink tab="tab-pe-systems" num="4" scrollId="desc-4"/>, the structure of PE2 is further enhanced by using human codon-optimized RT, a new linker containing a bipartite SV40 nuclear localization sequence (NLS)<TabScrollLink tab="tab-pe-systems" num="6" scrollId="desc-6"/>, an additional C-terminal c-Myc NLS<TabScrollLink tab="tab-pe-systems" num="7" scrollId="desc-7"/> and R221K N394K mutations in SpCas9 previously shown to improve Cas9 nuclease activity<TabScrollLink tab="tab-pe-systems" num="8" scrollId="desc-8"/>. These changes led to moderate improvements in editing efficiency compared to previous editor architectures.
+ With <b>PEmax</b><TabScrollLink tab="tab-pe-systems" num="8" scrollId="desc-8"/>{/* ehem 4 */}, the structure of PE2 is further enhanced by using human codon-optimized RT, a new linker containing a bipartite SV40 nuclear localization sequence (NLS)<TabScrollLink tab="tab-pe-systems" num="11" scrollId="desc-11"/>, an additional C-terminal c-Myc NLS<TabScrollLink tab="tab-pe-systems" num="12" scrollId="desc-12"/> and R221K N394K mutations in SpCas9 previously shown to improve Cas9 nuclease activity<TabScrollLink tab="tab-pe-systems" num="13" scrollId="desc-13"/>. These changes led to moderate improvements in editing efficiency compared to previous editor architectures.
</p>
</div>
<div className='col-4'>
@@ -416,7 +419,7 @@ export function Engineering() {
<div className='row align-items-center'>
<div className='col'>
<p>
- <b>PE6</b><TabScrollLink tab="tab-pe-systems" num="9" scrollId="desc-9"/> was made by improving the reverse transcriptase domain of the prime editor using Phage-Assisted Continuous Evolution (PACE). Multiple RT mutants (PE6a-d), derived from RTs of Escherichia coli Ec48 retron, Schizosaccharomyces pombe Tf1 retrotransposon and Moloney Murine Leukaemia Virus, were identified to increase editing efficiency over and/or were smaller than the M-MLV RT used in previous PE systems. Especially <b>PE6c</b> (evolved Tf1 RT) and <b>PE6d</b> (evolved M-MLV RT) showed significantly higher editing efficiencies than even PEmax depending on the targeted loci, with PE6d showing benefits especially in loci forming more complex secondary structures. Recent advancements in prime editing targeting the CFTR F508Δ mutation showed that PE6c was the most promising for editing in this loci<TabScrollLink tab="tab-pe-systems" num="10" scrollId="desc-10"/>. Improvements of nCas9 on the other hand (PE6e-g) were only marginal and highly site specific. All PE6 systems use nicking gRNAs (PE3) by default, but do not co-express MLH1dn.
+ <b>PE6</b><TabScrollLink tab="tab-pe-systems" num="14" scrollId="desc-14"/> was made by improving the reverse transcriptase domain of the prime editor using Phage-Assisted Continuous Evolution (PACE). Multiple RT mutants (PE6a-d), derived from RTs of Escherichia coli Ec48 retron, Schizosaccharomyces pombe Tf1 retrotransposon and Moloney Murine Leukaemia Virus, were identified to increase editing efficiency over and/or were smaller than the M-MLV RT used in previous PE systems. Especially <b>PE6c</b> (evolved Tf1 RT) and <b>PE6d</b> (evolved M-MLV RT) showed significantly higher editing efficiencies than even PEmax depending on the targeted loci, with PE6d showing benefits especially in loci forming more complex secondary structures. Recent advancements in prime editing targeting the CFTR F508Δ mutation showed that PE6c was the most promising for editing in this loci<TabScrollLink tab="tab-pe-systems" num="15" scrollId="desc-15"/>. Improvements of nCas9 on the other hand (PE6e-g) were only marginal and highly site specific. All PE6 systems use nicking gRNAs (PE3) by default, but do not co-express MLH1dn.
</p>
</div>
<div className='col-4'>
@@ -426,7 +429,7 @@ export function Engineering() {
<div className='row align-items-center'>
<div className='col'>
<p>
- <b>PE7</b><TabScrollLink tab="tab-pe-systems" num="11" scrollId="desc-11"/> adds an additional RNA binding domain to the Prime Editor. The domain is derived from the La Protein (La(1-194)), an endogenous eukaryotic protein involved RNA metabolism and known for its role in binding polyuridine (polyU) tails at the 3’ ends of nascent transcripts, thus protecting them from exonuclease activity. PE7 showed considerable improvements over PEmax at different loci and different types of edits when used with the PE2 strategy (no nicking gRNAs, no MLH1dn co-expression). Notably, PE7 did perform worse when used with engineered pegRNAs than with regular ones (see pegRNA design).
+ <b>PE7</b><TabScrollLink tab="tab-pe-systems" num="16" scrollId="desc-16"/> adds an additional RNA binding domain to the Prime Editor. The domain is derived from the La Protein (La(1-194)), an endogenous eukaryotic protein involved RNA metabolism and known for its role in binding polyuridine (polyU) tails at the 3’ ends of nascent transcripts, thus protecting them from exonuclease activity. PE7 showed considerable improvements over PEmax at different loci and different types of edits when used with the PE2 strategy (no nicking gRNAs, no MLH1dn co-expression). Notably, PE7 did perform worse when used with engineered pegRNAs than with regular ones (see pegRNA design).
</p>
</div>
<div className='col-4'>
@@ -449,7 +452,7 @@ export function Engineering() {
</p>
<H4 text="Test" id="text"/>
<p>
- To compare the prime editing performances of M-MLV RT (PE2) and PE<sup>CO</sup>-Mini RT, both were tested using a 2in1 prime editing reporter plasmid system<TabScrollLink tab="tab-pe-systems" num="12" scrollId="desc-12"/> (see <a onClick={() => goToPageWithTabAndScroll({scrollToId: 'Proof of Concept', path: '/engineering', tabId: 'tab-transfection' })}>Proof of Concept</a>) in HEK293 cells. Contrary to the findings of Gao et al., here the PE<sup>CO</sup>-Mini prime editor performed a lot worse than the PE2 prime editor.
+ To compare the prime editing performances of M-MLV RT (PE2) and PE<sup>CO</sup>-Mini RT, both were tested using a 2in1 prime editing reporter plasmid system<TabScrollLink tab="tab-pe-systems" num="17" scrollId="desc-17"/> (see <a onClick={() => goToPageWithTabAndScroll({scrollToId: 'Proof of Concept', path: '/engineering', tabId: 'tab-transfection' })}>Proof of Concept</a>) in HEK293 cells. Contrary to the findings of Gao et al., here the PE<sup>CO</sup>-Mini prime editor performed a lot worse than the PE2 prime editor.
</p>
<H4 text="Learn" id="text"/>
<p>
@@ -483,14 +486,14 @@ export function Engineering() {
<H3 text="PreCyse Casette" id="pe3head"/>
<H4 text="Design" id="text"/>
<p>
- In the later stages of our project, the Liu laboratory published their own findings regarding CFTR F508del targeting with prime editing<TabScrollLink tab="tab-pe-systems" num="10" scrollId="desc-10"/>. The data showed that the editing efficiency of PE2 based systems, even when using PE6c reverse transcriptase, might not be sufficient for application in a therapy. Also, the plasmids of current prime editors did not include restriction sites that would have allowed replacing components like the nickase to test alternatives. This is why, in a cherry-picking manner, we combined the PE6c architecture prime editor with the most promising aspects of other prime editors, creating the PreCyse cassette.
+ In the later stages of our project, the Liu laboratory published their own findings regarding CFTR F508del targeting with prime editing<TabScrollLink tab="tab-pe-systems" num="15" scrollId="desc-15"/>. The data showed that the editing efficiency of PE2 based systems, even when using PE6c reverse transcriptase, might not be sufficient for application in a therapy. Also, the plasmids of current prime editors did not include restriction sites that would have allowed replacing components like the nickase to test alternatives. This is why, in a cherry-picking manner, we combined the PE6c architecture prime editor with the most promising aspects of other prime editors, creating the PreCyse cassette.
</p>
<p>
Our decision on what components of existing prime editors we wanted to use was mainly driven by two factors: efficiency and precision. In prime editing, these two are often opposing forces, which means advancements improving efficiency often also increase the risk of off-targets mutations and on-target undesired editing. For this reason, we decided against using nicking gRNAs. Although they have been proven to reliably improve editing efficiency, they increase the risk and possible scope of off-target cleavage and mutations. Additionally, if <b>PE3b</b> is not applicable, there is a chance for double strand breaks to occur, which diminishes the safety advantage of prime editing over other common CRISPR-based methods. Co-expression of MLH1dn can improve editing efficiency in the same way as nicking gRNAs do, by helping to evade of the cellular mismatch repair mechanisms. The use of MLH1dn is especially impactful, when nicking gRNAs are not used, which is perfect in our case. Recently, the La poly(U)-binding motif has been shown to enhance prime editing efficiency, presumably through protection of the 3’ poly(U) tail of the pegRNA from RNases. The motif is also comparatively small, which aligns with the overall goal to create a compact prime editing tool. This is why PreCyse Casettes have been designed to include the La RNA binding motif fusion and the dominant negative MLH1 protein.
</p>
<H4 text="Build" id="text"/>
<p>
- The PreCyse cassette comes in three versions: PreCyseA, the most basic version, comprises of a T7 promoter and an open reading frame, which includes NLS and one typeIIS restriction enzyme cloning site for a nickase and a reverse transcriptase each. For possible future additions like e. g. selection markers, a BamHI restriction site at the end of the coding sequence allows for easy in-frame Gibson cloning. Building on this basis, PreCyseB expands PreCyseA by the La Poly(U)-binding motif. PreCyseC additionally introduces the co-expressed MLH1dn. The cassettes were ordered in three individual parts to be put together with a pCMV-PE6c backbone via Gibson Cloning in different configurations to create the three variants. In the plasmid the cassette is expressed under a CMV promoter and followed by a polyadenylation signal. The PreCyse Casettes themselves can be used as a BioBrick RFC[10] standard compatible composite part can thus be freely combined with other parts. The nickase and RT slots can be used for inserting any basic or composite part compatible with the Type IIS RCF[1000] standard for fusion proteins. The PreCyse Casette is meant to be a contribution to the iGEM community and a base for other teams to join us and researchers around the world to innovate in the exciting field of prime editing.
+ The PreCyse cassette comes in three versions: PreCyseA, the most basic version, comprises of a T7 promoter and an open reading frame, which includes NLS and one typeIIS restriction enzyme cloning site for a nickase and a reverse transcriptase each. For possible future additions like e. g. selection markers, a BamHI restriction site at the end of the coding sequence allows for easy in-frame Gibson cloning. Building on this basis, PreCyseB expands PreCyseA by the La Poly(U)-binding motif. PreCyseC additionally introduces the co-expressed MLH1dn. The cassettes were ordered in three individual parts to be put together with a pCMV-PE6c backbone via Gibson Cloning in different configurations to create the three variants. In the plasmid the cassette is expressed under a CMV promoter and followed by a polyadenylation signal. The PreCyse Casettes themselves can be used as a BioBrick RFC<TabScrollLink tab="tab-pe-systems" num="16" scrollId="desc-16"/> standard compatible composite part can thus be freely combined with other parts. The nickase and RT slots can be used for inserting any basic or composite part compatible with the Type IIS RCF[1000] standard for fusion proteins. The PreCyse Casette is meant to be a contribution to the iGEM community and a base for other teams to join us and researchers around the world to innovate in the exciting field of prime editing.
</p>
<div className="casettecontainer">
<div className="casettebox">
diff --git a/src/sources/eng-pe-sources.tsx b/src/sources/eng-pe-sources.tsx
index 2a19191d3e902a7d166418ad7ee2d7eb1d730910..69940a232dc0bca9deb623928751b8a0f9bc3bcd 100644
--- a/src/sources/eng-pe-sources.tsx
+++ b/src/sources/eng-pe-sources.tsx
@@ -3,13 +3,13 @@ import BibtexParser from "../components/makeSources";
export default function EngPEsystems(){
return (
<div>
- <BibtexParser bibtexSources={bibtexSources} />
+ <BibtexParser bibtexSources={bibtexSources} start={6}/>
</div>
);
}
-const bibtexSources = [
+const bibtexSources = [ /* 1-> 6 */
`@article{Anzalone_Randolph_Davis_Sousa_Koblan_Levy_Chen_Wilson_Newby_Raguram_2019,
title = {Search-and-replace genome editing without double-strand breaks or donor DNA},
author = {Anzalone, Andrew V. and Randolph, Peyton B. and Davis, Jessie R. and Sousa, Alexander A. and Koblan, Luke W. and Levy, Jonathan M. and Chen, Peter J. and Wilson, Christopher and Newby, Gregory A. and Raguram, Aditya and Liu, David R.},
@@ -26,6 +26,7 @@ const bibtexSources = [
abstractnote = {Most genetic variants that contribute to disease1 are challenging to correct efficiently and without excess byproducts2–5. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells, including targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates. We used prime editing in human cells to correct, efficiently and with few byproducts, the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay–Sachs disease (requiring a deletion in HEXA); to install a protective transversion in PRNP; and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homology-directed repair, has complementary strengths and weaknesses compared to base editing, and induces much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct up to 89% of known genetic variants associated with human diseases.},
language = {en}
}`,
+ /* 2 -> 7 Ehemals 7, jetzt 13 */
`@article{Jinek_Chylinski_Fonfara_Hauer_Doudna_Charpentier_2012,
title = {A programmable dual RNA-guided DNA endonuclease in adaptive bacterial immunity},
author = {Jinek, Martin and Chylinski, Krzysztof and Fonfara, Ines and Hauer, Michael and Doudna, Jennifer A. and Charpentier, Emmanuelle},
@@ -39,6 +40,7 @@ const bibtexSources = [
issn = {0036-8075},
abstractnote = {CRISPR/Cas systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using crRNAs to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA base-paired to trans-activating tracrRNA forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand while the Cas9 RuvC-like domain cleaves the non-complementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing., A two-RNA structure directs an endonuclease to cleave target DNA.}
}`,
+ /* 3 -> 8 Ehemals 8, jetzt 14 */
`@article{Gao_Ravendran_Mikkelsen_Haldrup_Cai_Ding_Paludan_Thomsen_Mikkelsen_Bak_2022,
title = {A truncated reverse transcriptase enhances prime editing by split AAV vectors},
author = {Gao, Zongliang and Ravendran, Sujan and Mikkelsen, Nanna S. and Haldrup, Jakob and Cai, Huiqiang and Ding, Xiangning and Paludan, Søren R. and Thomsen, Martin K. and Mikkelsen, Jacob Giehm and Bak, Rasmus O.},
@@ -53,6 +55,7 @@ const bibtexSources = [
issn = {1525-0016, 1525-0024},
language = {English}
}`,
+ /* 4 -> 9 Hemelas 9, jetzt 15 */
`@article{Chen_Hussmann_Yan_Knipping_Ravisankar_Chen_Chen_Nelson_Newby_Sahin_2021,
title = {Enhanced prime editing systems by manipulating cellular determinants of editing outcomes},
author = {Chen, Peter J. and Hussmann, Jeffrey A. and Yan, Jun and Knipping, Friederike and Ravisankar, Purnima and Chen, Pin-Fang and Chen, Cidi and Nelson, James W. and Newby, Gregory A. and Sahin, Mustafa and Osborn, Mark J. and Weissman, Jonathan S. and Adamson, Britt and Liu, David R.},
@@ -66,6 +69,7 @@ const bibtexSources = [
issn = {0092-8674},
abstractnote = {While prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types.}
}`,
+ /* 5 -> 10 Ehemals 10, jetzt 16 */
`@article{Tomer_Buermeyer_Nguyen_Liskay_2002,
title = {Contribution of Human Mlh1 and Pms2 ATPase Activities to DNA Mismatch Repair},
author = {Tomer, Guy and Buermeyer, Andrew B. and Nguyen, Megan M. and Liskay, R. Michael},
@@ -79,6 +83,7 @@ const bibtexSources = [
issn = {0021-9258},
abstractnote = {MutLα, a heterodimer composed of Mlh1 and Pms2, is the major MutL activity in mammalian DNA mismatch repair. Highly conserved motifs in the N termini of both subunits predict that the protein is an ATPase. To study the significance of these motifs to mismatch repair, we have expressed in insect cells wild type human MutLα and forms altered in conserved glutamic acid residues, predicted to catalyze ATP hydrolysis of Mlh1, Pms2, or both. Using an in vitro assay, we showed that MutLα proteins altered in either glutamic acid residue were each partially defective in mismatch repair, whereas the double mutant showed no detectable mismatch repair. Neither strand specificity nor directionality of repair was affected in the single mutant proteins. Limited proteolysis studies of MutLα demonstrated that both Mlh1 and Pms2 N-terminal domains undergo ATP-induced conformational changes, but the extent of the conformational change for Mlh1 was more apparent than for Pms2. Furthermore, Mlh1 was protected at lower ATP concentrations than Pms2, suggesting Mlh1 binds ATP with higher affinity. These findings imply that ATP hydrolysis is required for MutLα activity in mismatch repair and that this activity is associated with differential conformational changes in Mlh1 and Pms2.}
}`,
+ /* 6 -> 11 Ehemals 11, jetzt 17 */
`@article{Wu_Corbett_Berland_2009,
title = {The Intracellular Mobility of Nuclear Import Receptors and NLS Cargoes},
author = {Wu, Jianrong and Corbett, Anita H. and Berland, Keith M.},
@@ -92,6 +97,7 @@ const bibtexSources = [
issn = {0006-3495},
abstractnote = {We have investigated classical nuclear localization sequence (NLS) mediated protein trafficking by measuring biomolecular dynamics within living cells using two-photon fluorescence correlation spectroscopy. By directly observing the behavior of specific molecules in their native cellular environment, it is possible to uncover functional details that are not apparent from traditional biochemical investigations or functional assays. We show that the intracellular mobility of NLS cargoes and their import receptor proteins, karyopherin-α and karyopherin-β, can be robustly measured and that quantitative comparison of intracellular diffusion coefficients provides new insights into nuclear transport mechanisms. Import cargo complexes are assembled throughout the cytoplasm, and their diffusion is slower than predicted by molecular weight due to specific interactions. Analysis of NLS cargo diffusion in the cytoplasm indicates that these interactions are likely disrupted by NLS cargo binding. Our results suggest that delivery of import receptors and NLS cargoes to nuclear pores may complement selective translocation through the pores as a functional mechanism for regulating transport of proteins into the nucleus.}
}`,
+ /* 7 -> 12 Ehemals 12, jetzt 18 */
`@article{Dang_Lee_1988,
title = {Identification of the human c-myc protein nuclear translocation signal},
author = {Dang, C.V. and Lee, W.M.F.},
@@ -101,7 +107,9 @@ const bibtexSources = [
number = 10,
pages = {4048–4054},
doi = {10.1128/MCB.8.10.4048}
- }`,
+ }`
+ ,
+ /* 8 -> 13 */
`@article{Spencer_Zhang_2017,
title = {Deep mutational scanning of S. pyogenes Cas9 reveals important functional domains},
author = {Spencer, J.M. and Zhang, X.},
@@ -111,8 +119,9 @@ const bibtexSources = [
number = 1,
doi = {10.1038/s41598-017-17081-y},
abstractnote = {RNA-guided endonucleases (RGENs) have invigorated the field of site-specific nucleases. The success of Streptococcus pyogenes Cas9 (SpCas9) has led to the discovery of several other CRISPR-Associated RGENs. As more RGENs become available, it will be necessary to refine their activity before they can be translated into the clinic. With this in mind, we sought to demonstrate how deep mutational scanning (DMS) could provide details about important functional regions in SpCas9 and speed engineering efforts. Consequently, we developed a nuclease screening platform which could distinguish active Cas9 mutants. We screened a library of 1.9 × 107 with over 8500 possible non-synonymous mutations and inferred the effects of each mutation using DMS. We demonstrate that the RuvC and HNH domains are the least tolerant regions to mutation. In contrast, the Rec2 and PI domains tolerate mutation better than other regions. The mutation information defined in this work provides a foundation for further SpCas9 engineering. Together, our results demonstrate how DMS can be a powerful tool to uncover features important to RGEN function. Application of this approach to emerging RGENs should enhance their engineering and optimization for therapeutic and other applications. © 2017 The Author(s).}
- }`,
- `@article{Doman_Pandey_Neugebauer_An_Davis_Randolph_McElroy_Gao_Raguram_Richter_2023,
+ }`,
+ /* 9 -> 14 */
+ `@article{Doman_Pandey_Neugebauer_An_Davis_Randolph_McElroy_Gao_Raguram_Richter_2023,
title = {Phage-assisted evolution and protein engineering yield compact, efficient prime editors},
author = {Doman, Jordan L. and Pandey, Smriti and Neugebauer, Monica E. and An, Meirui and Davis, Jessie R. and Randolph, Peyton B. and McElroy, Amber and Gao, Xin D. and Raguram, Aditya and Richter, Michelle F. and Everette, Kelcee A. and Banskota, Samagya and Tian, Kathryn and Tao, Y. Allen and Tolar, Jakub and Osborn, Mark J. and Liu, David R.},
year = 2023,
@@ -125,7 +134,7 @@ const bibtexSources = [
doi = {10.1016/j.cell.2023.07.039},
issn = {0092-8674, 1097-4172},
language = {English}
- }`,
+ }`, /* 10 -> 15 */
`@article{Sousa_Hemez_Lei_Traore_Kulhankova_Newby_Doman_Oye_Pandey_Karp_2024,
title = {Systematic optimization of prime editing for the efficient functional correction of CFTR F508del in human airway epithelial cells},
author = {Sousa, Alexander A. and Hemez, Colin and Lei, Lei and Traore, Soumba and Kulhankova, Katarina and Newby, Gregory A. and Doman, Jordan L. and Oye, Keyede and Pandey, Smriti and Karp, Philip H. and McCray, Paul B. and Liu, David R.},
@@ -139,7 +148,8 @@ const bibtexSources = [
rights = {2024 The Author(s)},
abstractnote = {Prime editing (PE) enables precise and versatile genome editing without requiring double-stranded DNA breaks. Here we describe the systematic optimization of PE systems to efficiently correct human Cystic Fibrosis (CF) transmembrane conductance regulator (CFTR) F508del, a three-nucleotide deletion that is the predominant cause of CF. By combining six efficiency optimizations for PE—engineered PE guide RNAs, the PEmax architecture, the transient expression of a dominant-negative mismatch repair protein, strategic silent edits, PE6 variants and proximal ‘dead’ single-guide RNAs—we increased correction efficiencies for CFTR F508del from less than 0.5% in HEK293T cells to 58% in immortalized bronchial epithelial cells (a 140-fold improvement) and to 25% in patient-derived airway epithelial cells. The optimizations also resulted in minimal off-target editing, in edit-to-indel ratios 3.5-fold greater than those achieved by nuclease-mediated homology-directed repair, and in the functional restoration of CFTR ion channels to over 50% of wild-type levels (similar to those achieved via combination treatment with elexacaftor, tezacaftor and ivacaftor) in primary airway cells. Our findings support the feasibility of a durable one-time treatment for CF.},
language = {en}
- }`,
+ }`,
+ /* 11 -> 16*/
`@article{Yan_Oyler-Castrillo_Ravisankar_Ward_Levesque_Jing_Simpson_Zhao_Li_Yan_2024,
title = {Improving prime editing with an endogenous small RNA-binding protein},
author = {Yan, Jun and Oyler-Castrillo, Paul and Ravisankar, Purnima and Ward, Carl C. and Levesque, Sébastien and Jing, Yangwode and Simpson, Danny and Zhao, Anqi and Li, Hui and Yan, Weihao and Goudy, Laine and Schmidt, Ralf and Solley, Sabrina C. and Gilbert, Luke A. and Chan, Michelle M. and Bauer, Daniel E. and Marson, Alexander and Parsons, Lance R. and Adamson, Britt},
@@ -153,7 +163,9 @@ const bibtexSources = [
issn = {0028-0836, 1476-4687},
abstractnote = {Abstract Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3′ ends of CRISPR–Cas guide RNAs 1 . To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3′ ends of RNA polymerase III transcripts 2 . We found that La functionally interacts with the 3′ ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.},
language = {en}
- }`,
+ }`,
+ /* 12 -> 17*/
+
`@article{Simon,
title = {PEAR, a flexible fluorescent reporter for the identification and enrichment of successfully prime edited cells},
author = {Simon, Dorottya Anna and Tálas, András and Kulcsár, Péter István and Biczók, Zsuzsanna and Krausz, Sarah Laura and Várady, György and Welker, Ervin},
@@ -166,5 +178,5 @@ const bibtexSources = [
issn = {2050-084X},
abstractnote = {Prime editing is a recently developed CRISPR/Cas9 based gene engineering tool that allows the introduction of short insertions, deletions, and substitutions into the genome. However, the efficiency of prime editing, which typically achieves editing rates of around 10%–30%, has not matched its versatility. Here, we introduce the prime editor activity reporter (PEAR), a sensitive fluorescent tool for identifying single cells with prime editing activity. PEAR has no background fluorescence and specifically indicates prime editing events. Its design provides apparently unlimited flexibility for sequence variation along the entire length of the spacer sequence, making it uniquely suited for systematic investigation of sequence features that influence prime editing activity. The use of PEAR as an enrichment marker for prime editing can increase the edited population by up to 84%, thus significantly improving the applicability of prime editing for basic research and biotechnological applications.},
editor = {Lapinaite, Audrone and Stainier, Didier YR and Hamilton, Jennifer R}
- }`
+ }`
]
\ No newline at end of file
diff --git a/src/sources/eng-trf-sources.tsx b/src/sources/eng-trf-sources.tsx
index 866091d438d9ce97a34834934c39f5507568638d..7f908860dadd62795878a5193fb7fb3abf18c672 100644
--- a/src/sources/eng-trf-sources.tsx
+++ b/src/sources/eng-trf-sources.tsx
@@ -3,7 +3,7 @@ import BibtexParser from "../components/makeSources";
export default function EngTrfsources(){
return (
<div>
- <BibtexParser bibtexSources={bibtexSources} />
+ <BibtexParser bibtexSources={bibtexSources} start={3} />
</div>
);
}