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Commit c17dc29e authored by Liliana Sanfilippo's avatar Liliana Sanfilippo
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Merge branch 'main' of ssh://gitlab.igem.org/2024/bielefeld-cebitec

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src/contents/engineering.tsx
+ 84
− 38
View file @ c17dc29e
......@@ -10,6 +10,8 @@ import { TabScrollLink } from "../components/Link";
import { InfoBox } from "../components/Boxes";
import { DownloadLink } from "../components/Buttons";
import EngPEsystems from "../sources/eng-pe-sources";
import EngPoCsources from "../sources/eng-poc-sources";
import { Section } from "../components/sections";
......@@ -45,63 +47,107 @@ export function Engineering() {
<section >
<div className="eng-box box" >
<H3 id="Proof of Concept" text="Proof of Concept"></H3>
<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[1]. Apart from easy handling and comparatively easy transfection, they have, as we found out in our exchange with Mattijs Bulcaen, 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[2] and optimizing transfection protocols.</p>
<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-proof-of-concept" num="1" scrollId="desc-1"/>. Apart from easy handling and comparatively easy transfection, they have, as we found out in our exchange with Mattijs Bulcaen[link], 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-proof-of-concept" num="2" scrollId="desc-2"/> and optimizing transfection protocols.
</p>
</div>
<div className="box" >
<p id="cyc1">
<H3 text="Test of Lipofectamine 2000" id="text"/>
<H4 text="Test" id="text"/>
<p>While conducting research on transfection methods for HEK cells, particular attention was devoted to the delivery of the Prime Editing complex into the cells. In the literature, Lipofectamine is described as a common transfection agent. <i>Anzalone et al. 2019</i>[3] describe a transfection of prime-editing complexes with Lipofectamine 2000. The aim of this study is to introduce our prime-editing complex into HEK cells.</p>
<p>Transfection with Lipofectamine 2000 was performed in accordance with the Anzalone protocol. However, the result was characterized by insufficient transfection efficiency.</p>
<H4 text="Learn" id="text"/>
<p>The low efficiency of Lipofectamine 2000 indicated that the product is not optimally suited to the specific conditions under consideration. In contrast, Lipofectamine 3000 is described in the literature as potentially more efficient.</p>
<H4 text="Design" id="text"/>
<p>In light of the aforementioned findings, the decision was taken to test Lipofectamine 3000, given its reputation for greater efficiency. A new test design was devised, utilizing Lipofectamine 3000 with an equivalent quantity of DNA and modified transfection conditions.</p>
<p>
Before testing any of our mechanistic approaches, we had to examine whether we can facilitate and detect prime editing in the first place. During our research we eventually stumbled upon the PEAR reporter system (see pegRNA engineering cycle[link]). The PEAR 2in1 plasmid reporter includes a GFP that is to be edited for sensitive prime editing detection, and a pegRNA expression cassette with a pegRNA targeting the plasmid itself. Having found a system capable of detecting even small-scale prime editing, the next step was to find transfection conditions that would work. In the literature, Lipofectamine is described as a common transfection agent.
</p>
<p>
Transfection with Lipofectamine 2000 was performed in accordance with the Anzalone protocol. However, the result was characterized by insufficient transfection efficiency.
</p>
<H4 text="Build" id="text"/>
<p>In accordance with the established protocol, the recommended ratio of 1 µg DNA to 2 µl Lipofectamine 3000, as provided by ThermoFisher, is to be employed.</p>
<p>
Anzalone et al. 2019<TabScrollLink tab="tab-proof-of-concept" num="3" scrollId="desc-3"/> describe a transfection of prime-editing complexes with Lipofectamine 2000.
</p>
<H4 text="Test" id="text"/>
<p>
Transfection with Lipofectamine 2000 was performed in accordance with the Anzalone protocol. However, the result was characterized by insufficient transfection efficiency.
</p>
<H4 text="Learn" id="text"/>
<p>
The low efficiency of Lipofectamine 2000 indicates that the product is not optimally suited to the specific conditions under consideration. In contrast, Lipofectamine 3000 is described in the literature as potentially more efficient.
</p>
</p>
</div>
<div className="box" >
<p id="cyc2">
<H3 text="Initial Test with Lipofectamine 3000" id="text"/>
<H4 text="Test" id="text"/>
<p>Considering the favorable assessment of Lipofectamine 3000 in the scientific literature, the proof of concept was conducted once more.</p>
<p>The objective of the experiment was to enhance the transfection efficiency of Lipofectamine 3000. The transfection protocol was conducted in accordance with the manufacturer's instructions (1 µg DNA, 2 µl Lipofectamine 3000 reagent).</p>
<p>The outcome revealed that despite the modification, the transfection efficiency remained inadequate, although a marginal improvement was discernible.</p>
<H4 text="Learn" id="text"/>
<p>Although a switch to Lipofectamine 3000 resulted in a marginal improvement, the efficiency fell short of expectations. This indicates that further optimization is required in terms of the amount of Lipofectamine and DNA, as well as the medium used.</p>
<H4 text="Design" id="text"/>
<p>In order to optimize the transfection process, a new optimization test was designed, which incorporates a variable design with regard to the quantity of Lipofectamine 3000 and DNA.</p>
<p>
In light of the aforementioned findings, the decision was taken to test Lipofectamine 3000, given its reputation for greater efficiency. A new test design was devised, utilizing Lipofectamine 3000 with an equivalent quantity of DNA and modified transfection conditions.
</p>
<H4 text="Build" id="text"/>
<p>The protocol entails the utilization of varying concentrations of Lipofectamine 3000, specifically 1 µl and 1.5 µl, with a DNA quantity of 1 µg or 0.5 µg.</p>
<p>
In accordance with the established protocol, the recommended ratio of 1 µg DNA to 2 µl Lipofectamine 3000, as provided by ThermoFisher, was to be employed.
</p>
<H4 text="Test" id="text"/>
<p>
The objective of the experiment was to enhance the transfection efficiency of Lipofectamine 3000. The transfection protocol was conducted in accordance with the manufacturer's instructions (1 µg DNA, 2 µl Lipofectamine 3000 reagent).
</p>
<p>
The outcome revealed that despite the modification, the transfection efficiency remained inadequate, although a marginal improvement was discernible.
</p>
<H4 text="Learn" id="text"/>
<p>
Although a switch to Lipofectamine 3000 resulted in a marginal improvement, the efficiency fell short of expectations. This indicates that further optimization is required in terms of the amount of Lipofectamine and DNA, as well as the medium used.
</p>
</p>
</div>
<div className="box" >
<p id="cyc3">
<H3 text="Optimization of DNA and Lipofectamine Volumes" id="text"/>
<H4 text="Test" id="text"/>
<p>To enhance transfection efficiency, optimization tests were conducted, in which the quantities of Lipofectamine and DNA were varied. The objective of this iteration was to find the optimal ratio of Lipofectamine 3000 to DNA. To this end, 1 µl and 1.5 µl of Lipofectamine 3000 at a DNA concentration of either 1 µg or 0.5 µg were compared with each other.</p>
<H4 text="Learn" id="text"/>
<p>The experiment demonstrated that a quantity of 1 µl Lipofectamine 3000 is sufficient for successful transfection, and that increasing the quantity does not result in a notable difference. Additionally, the findings indicated that an amount of 1 µg DNA exhibited a higher efficiency than an amount of 0.5 µg DNA. It can be reasoned that additional factors may have contributed to the previously observed decline in transfection efficiency. One potential explanation is that the cells may have been in an excessively high passage level.</p>
<p>It can be reasonably deduced that the aforementioned factors may have contributed to the observed decline in transfection efficiency.</p>
<H4 text="Design" id="text"/>
<p>The results obtained were used to develop an optimized protocol that takes into account both the concentration of Lipofectamine and the amount of DNA.</p>
<p>
In order to optimize the transfection process, a new optimization test was designed, which incorporated a variable design with regard to the quantity of Lipofectamine 3000 and DNA.
</p>
<H4 text="Build" id="text"/>
<p>In the following experiments, a DNA quantity of 1 µg and a defined quantity of 1 µl Lipofectamine 3000 was used.</p>
<p>
The protocol entailed the utilization of varying concentrations of Lipofectamine 3000, specifically 1 µl and 1.5 µl, with a DNA quantity of 1 µg or 0.5 µg.
</p>
<H4 text="Test" id="text"/>
<p>
To enhance transfection efficiency, optimization tests were conducted, in which the quantities of Lipofectamine and DNA were varied. The objective of this iteration was to find the optimal ratio of Lipofectamine 3000 to DNA. To this end, 1 µl and 1.5 µl of Lipofectamine 3000 at a DNA concentration of either 1 µg or 0.5 µg were compared with each other.
</p>
<H4 text="Learn" id="text"/>
<p>
The experiment demonstrated that a quantity of 1 µl Lipofectamine 3000 was sufficient for successful transfection, and that increasing the quantity does not result in a notable difference. Additionally, the findings indicated that an amount of 1 µg DNA exhibited a higher efficiency than an amount of 0.5 µg DNA. It can be reasoned that additional factors may have contributed to the previously observed decline in transfection efficiency. One potential explanation is that the cells may have been in an excessively high passage level.
</p>
<p>
It can be reasonably deduced that the aforementioned factors may have contributed to the observed decline in transfection efficiency.
</p>
</p>
</div>
<div className="box" >
<p id="cyc4">
<H3 text="Validation of optimized Protocol" id="text"/>
<H4 text="Design" id="text"/>
<p>
The results obtained were used to develop an optimized protocol that takes into account both the concentration of Lipofectamine and the amount of DNA.
</p>
<H4 text="Buld" id="text"/>
<p>
In subsequent research, a DNA quantity of 1 µg and a defined quantity of 1 µl of Lipofectamine 3000 will be utilized.
</p>
<H4 text="Test" id="text"/>
<p>Following the series of optimizations, the proof of concept was conducted once more to confirm the efficacy of the optimized protocol. This protocol involved the utilization of 1 µl Lipofectamine 3000, 1 µg DNA, 2 µl Reagent 3000 and Opti-MEM as a medium. The outcomes were encouraging, as the transfection efficiency was markedly enhanced.</p>
<p>
Following a series of optimizations, the proof of concept was conducted once more to confirm the efficacy of the optimized protocol. The objective was to perform the transfection with the final, optimized protocol. This protocol involved the utilization of 1 µl Lipofectamine 3000, 1 µg DNA, 2 µl Reagent 3000 and Opti-MEM as a medium. The outcomes were encouraging, as the transfection efficiency was markedly enhanced.
</p>
<H4 text="Learn" id="text"/>
<p>The utilization of an optimized quantity of 1 µl Lipofectamine 3000, a defined quantity of DNA and the suitable Opti-MEM medium resulted in a notable enhancement in transfection efficiency. This substantiates the assertion that the aforementioned conditions constitute an optimal foundation for the transfection of HEK cells with the prime editing complex.</p>
<H4 text="Design" id="text"/>
<p>This protocol establishes the standard procedure for the transfection of HEK cells with the Prime Editing Complex. The transfection reagent Lipofectamine 3000 is diluted in Opti-MEM to a final volume of 1 µl, and the DNA to be transfected is diluted to a final concentration of 1 µl.</p>
<p>
The utilization of an optimized quantity of 1 µl Lipofectamine 3000, a defined quantity of DNA and the suitable Opti-MEM medium resulted in a notable enhancement in transfection efficiency. This substantiates the assertion that the aforementioned conditions constitute an optimal foundation for the transfection of HEK cells with the prime editing complex.
</p>
</p>
</div>
<Section title="References" id="references">
<EngPoCsources/>
</Section>
<br/>
<div className="row ">
<div className="col">
......@@ -130,10 +176,10 @@ export function Engineering() {
<div className='row align-items-center'>
<div className='col'>
<p>
<b>PE1</b>[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[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="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.
</p>
<p>
<b>PE2</b>[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)[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="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.
</p>
</div>
<div className='col-4'>
......@@ -143,7 +189,7 @@ export function Engineering() {
<div className='row align-items-center'>
<div className='col'>
<p>
The <b>PE3</b>[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="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.
</p>
</div>
<div className='col-4'>
......@@ -153,10 +199,10 @@ export function Engineering() {
<div className='row align-items-center'>
<div className='col'>
<p>
<b>PE4</b> and <b>PE5</b>[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[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="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.
</p>
<p>
With <b>PEmax</b>[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)[[6]], an additional C-terminal c-Myc NLS[7] and R221K N394K mutations in SpCas9 previously shown to improve Cas9 nuclease activity[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="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.
</p>
</div>
<div className='col-4'>
......@@ -167,7 +213,7 @@ export function Engineering() {
<div className='row align-items-center'>
<div className='col'>
<p>
<b>PE6</b>[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[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="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.
</p>
</div>
<div className='col-4'>
......@@ -177,7 +223,7 @@ export function Engineering() {
<div className='row align-items-center'>
<div className='col'>
<p>
<b>PE7</b>[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="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).
</p>
</div>
<div className='col-4'>
......@@ -200,7 +246,7 @@ 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[12] (see Prove of Concept[link]) 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="12" scrollId="desc-12"/> (see Prove of Concept[link]) 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>
......@@ -234,7 +280,7 @@ export function Engineering() {
<H3 text="PreCyse Casette" id="text"/>
<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[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="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.
</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.
......@@ -263,9 +309,9 @@ export function Engineering() {
<p></p> */}
</p>
</div>
<section title="references" id="references">
<Section title="References" id="references">
<EngPEsystems/>
</section>
</Section>
<br/>
<div className="row ">
<div className="col">
......
src/sources/eng-pe-sources.tsx
+ 1
− 1
View file @ c17dc29e
......@@ -154,7 +154,7 @@ const bibtexSources = [
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}
}`,
`@article{Simon_Tálas_Kulcsár_Biczók_Krausz_Várady_Welker_2022,
`@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},
year = 2022,
......
src/sources/eng-poc-sources.tsx 0 → 100644
+ 56
− 0
View file @ c17dc29e
import BibtexParser from "../components/makeSources";
export default function EngPoCsources(){
return (
<div>
<BibtexParser bibtexSources={bibtexSources} />
</div>
);
}
const bibtexSources = [
`@article{Graham_Smiley_Russell_Nairn_1977,
title = {Characteristics of a Human Cell Line Transformed by DNA from Human Adenovirus Type 5},
author = {Graham, F. L. and Smiley, J. and Russell, W. C. and Nairn, R.},
year = 1977,
journal = {Journal of General Virology},
publisher = {Microbiology Society,},
volume = 36,
number = 1,
pages = {59–72},
doi = {10.1099/0022-1317-36-1-59},
issn = {1465-2099},
abstractnote = {SUMMARY Human embryonic kidney cells have been transformed by exposing cells to sheared fragments of adenovirus type 5 DNA. The transformed cells (designated 293 cells) exhibited many of the characteristics of transformation including the elaboration of a virus-specific tumour antigen. Analysis of the polypeptides synthesized in the 293 cells by labelling with 35S-methionine and SDS PAGE showed a variable pattern of synthesis, different in a number of respects from that seen in other human cells. On labelling the surface of cells by lactoperoxidase catalysed radio-iodination, the absence of a labelled polypeptide analogous to the 250 K (LETS) glycoprotein was noted. Hybridization of labelled cellular RNA with restriction fragments of adenovirus type 5 DNA indicated transcription of a portion of the adenovirus genome at the conventional left hand end.}
}`,
`@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},
year = 2022,
month = feb,
journal = {eLife},
publisher = {eLife Sciences Publications, Ltd},
volume = 11,
pages = {e69504},
doi = {10.7554/eLife.69504},
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}
}`,
`@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.},
year = 2019,
month = dec,
journal = {Nature},
publisher = {Nature Publishing Group},
volume = 576,
number = 7785,
pages = {149–157},
doi = {10.1038/s41586-019-1711-4},
issn = {1476-4687},
rights = {2019 The Author(s), under exclusive licence to Springer Nature Limited},
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}
}`
]
\ No newline at end of file
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