diff --git a/src/contents/engineering.tsx b/src/contents/engineering.tsx
index 4002aa8998bbc58f2be311f4c617ab0eca289b0c..b219c03be18dc5732f64fa82ea0a4a531432b44b 100644
--- a/src/contents/engineering.tsx
+++ b/src/contents/engineering.tsx
@@ -408,11 +408,11 @@ export function Engineering() {
</p>
<H4 text="Build" id="text"/>
<p>
- We designed primers for the amplification of PE<sup>CO</sup>-Mini RT and cloned it into pCMV-PE2 via double digestion and Gibson assembly.
+ We designed primers for the amplification of PE<sup>CO</sup>-Mini RT and cloned it into pCMV-PE2 via double digestion and Gibson assembly.
</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="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.
</p>
<H4 text="Learn" id="text"/>
<p>
@@ -504,110 +504,146 @@ export function Engineering() {
<section id="pegRNA sec" >
<div className="eng-box box" >
<H2 id="pegrna-header" text="pegRNA"></H2>
- <p><LoremShort></LoremShort></p>
+ <p>The pegRNA[link] is of paramount importance for function and efficiency of prime editors, as it plays a role in every step of the prime editing mechanism. It is therefore equally important to optimize the pegRNA than it is to have an optimized prime editor. Hence this engineering cycle explains our process of optimizing the pegRNAs for our genomic target, CFTR F508del. Given that different areas of the pegRNA have different functionalities, the following iteration cycles will demonstrate how improvements and optimizations have been made to these various functional domains in relation to the CFTR context. This was achieved through research, the correspondence with of experts and experiments.</p>
</div>
<div className="box" >
<p id="peg1">
<H3 text="peg1" id="peg1head"/>
+ <p>
+ The first iteration of our engineering cycle, we designed our first set of pegRNAs targeting the modified PEAR reporter[link]. focused on the incorporation of silent edits into the reverse transcriptase template.
+ </p>
<H4 text="Design" id="design-head"/>
<p>
-
+ Following an interview with Jan-Phillipp Gerhard[link], we came across the concept of silent edits. Silent edits refer to single-base alterations of the nucleotide sequence that do not change the encoded amino acid. Jan-Phillipp pointed out that introducing silent edits in addition to the intended edit offers two major advantages.
+ </p>
+ <p>
+ Firstly, silent edits can increase the likelihood of flap incorporation during the prime editing process, especially in the context of MMR (Mismatch Repair) in the cell. Without silent edits, the cell is more likely to detect the mismatches that only occur at the desired mutation site, leading to a higher chance of the wild-type flap being reinserted. By introducing silent edits, multiple mismatches are present which this increases the probability of the synthesized flap being incorporated.
+ </p>
+ <p>
+ Secondly, silent edits can prevent re-binding of the prime editing complex to the target region after successful editing. This is be achieved by introducing silent edits to the regions making up PAM sequence and/or protospacer. PAM or protospacer disruption make the editing process more secure. This is because it reduces the likelihood of editing the target region repeatedly, which would increase the probability of on-target undesired editing outcomes. He suggested that swapping cytosine or guanine bases for these silent edits can be particularly effective in improving prime editing efficiency.
</p>
<H4 text="Build" id="build-head"/>
<p>
-
+ We designed several pegRNAs, both with and without silent edits. To assist with this, we used the pegFinder software<TabScrollLink tab="tab-pegrna" num="1" scrollId="desc-1"/>, which generated possible variations of pegRNAs based on the sequence of the reporter plasmid. We selected the optimal pegRNA as suggested by the software, and then tested it in two forms: one unmodified and one with silent edits. For the unmodified variant, we included a single silent edit that introduced a PAM disrupt in terms of our biosafety measures. For the modified variant, we introduced three silent edits in total, adding two more to the initial edit.
+ </p>
+ <p>
+ Once we had designed these variants, we ordered them in their individual components and cloned them into a pU6-peg-GG-acceptor backbone using Golden Gate cloning according to the protocol from Anzalone et al. 2019<TabScrollLink tab="tab-pegrna" num="2" scrollId="desc-2"/>. We then screened the assembled pegRNAs to ensure that the individual components had the correct orientation and then cloned them into the pU6-GG-pegRNA-acceptor plasmid so that they were ready to be tested.
</p>
<H4 text="Test" id="test-head"/>
<p>
-
+ These two variants were then tested against each other using our reporter plasmid system[link] and a PE2 prime editor[link]. The test of the pegRNAs was conducted by co-transfecting the reporter system, the pegRNA plasmids and the PE2 plasmids into HEK293 cells.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
-
+ The results showed that the editing efficiency of the variant without silent edits was superior to the variant with silent edits, which considering our input was not expected. But as we have learned in the interview with Jan-Phillipp Gerhard, these silent edits are especially effective in avoiding mismatch repair (MMR) inside human cells. Form Mattijs Bulcaen[link] we learned, that HEK293 cells are deficient in this very mechanism. From this we deduced that we had to test the silent edits in lung ephital cells to get a valid result.
</p>
</p>
</div>
<div className="box" >
<p id="peg2">
<H3 text="peg2" id="peg2head"/>
+ <p>
+ In this second iteration, we focused on further optimizing our pegRNA by incorporating a stem loop and experimenting with different lengths of the PBS (Primer Binding Site) and RTT (Reverse Transcriptase Template). These modifications were inspired by a combination of literature research and expert interviews. After evaluating the performance of the pegRNAs using FACS, we selected the three most effective candidates.
+ </p>
<H4 text="Design" id="design-head"/>
<p>
-
+ Based on literature reviews and our interview with Mattijs Bulcaen[link], we decided to modify our pegRNA by adding a stem loop to enhance its stability. Specifically, Mattijs recommended using the tevopreQ1 stem loop, a small structural motif that increases the pegRNA's resistance to RNases. This stem loop was added to the 3' end of the pegRNA, positioned after the PBS.
+ </p>
+ <p>
+ Additionally, during a webinar with B. Sc. Jordan Doman ([link]https://www.youtube.com/watch?v=0Z_ztvkvKUA), we learned that it is important to test various lengths of PBS and RTT, as there is no universally optimal length for all applications. Instead, the ideal lengths are application specific. Following this advice, we designed six different pegRNA variants with combinations of two different PBS lengths (16 and 17 nucleotides) and three different RTT lengths (27, 30, and 33 nucleotides).
+ </p>
+ <p>
+ We chose the PBS lengths of 16 and 17 nucleotides based on an earlier recommendation from Jan-Phillipp Gerhard, who emphasised that the annealing temperature of the PBS should match the environmental conditions relevant to the intended application. In our case, since we are exploring a potential therapeutic approach, it is important that the annealing temperature of the PBS is close to the body temperature of 37 °C, which is the case for these lengths. The RTT lengths were selected based on suggestions from the pegFinder software. As with our previous insights, we designed all six variants both with and without silent edits for a wider comparison of the silent edits, making it 12 variants in total.
</p>
<H4 text="Build" id="build-head"/>
<p>
-
+ Once we had designed these variants, we ordered them in their individual components and cloned them together using Golden Gate cloning. This was a much more resource-efficient and sustainable option, as only the PBS and/or RTT lengths differed. Thus, there was a constant pegRNA part, consisting of spacer and scaffold, and a variable part, consisting of PBS, RTT and stem loop. We then cloned these variants into the pU6-GG-pegRNA-acceptor plasmid and confirmed the correct orientation and successful cloning of all constructs through screening.
</p>
<H4 text="Test" id="test-head"/>
<p>
-
+ We tested these twelve pegRNA variants against each other and the two previous variants without the trevopreQ1 stem loop, again within the PE2 system, using our reporter system, to assess their editing efficiency. The experimental setup was similar to the cycle before.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
-
+ From this round of testing, we found out that our engineered pegRNA variants pegRNA04, 05, 07 and 08 exhibited the highest levels of efficiency and stability, while the pegRNA12 showed the lowest level of editing efficiency. Therefore, we reasoned to go with these four pegRNA variants as well as pegRNA12 as a negative example for follow-up experiments.
</p>
</p>
</div>
<div className="box" >
<p id="peg3">
<H3 text="peg3" id="peg3head"/>
+ <p>
+ HEK cells are an easy to handle and easy to edit cell model. However, they are not particularly similar to the cells that would actually be useful targets for a gene therapy. In our context, two key differences are especially grave: HEK cells, as mentioned above, are impaired in mismatch repair, making them easier to edit, and they do not naturally express CFTR.
+ </p>
<H4 text="Design" id="design-head"/>
<p>
-
+ In this third iteration, we wanted to investigatee the applicability of a pegRNA optimized in a model closer to therapeutic application. In our case we used in CFBE41o- epithelial cells lines[link] homozygous for the CFTR F508del mutation.
</p>
<H4 text="Build" id="build-head"/>
<p>
-
+ For this test, we used one of the pegRNAs (pegRNA04) that showed the highest efficiencies in previous optimization steps. Since we expected only low editing efficiencies compared to HEK cells for reasons mentioned above, we used the PE6c prime editor[link]. It had proven to be most effective in HEK cells in our pe systems engineering cycle[link] and should ensure detectability of possible editing.
</p>
<H4 text="Test" id="test-head"/>
<p>
-
+ We co-transfected the CFBE41o- with our modified reporter plasmid[link], the plasmid expressing pegRNA04 as well as pCMV-PE6c. As a result, we observed fluorescence, indicating successful editing of the reporter plasmid. The negative controls transfected with only one of the plasmids each showed no fluorescence, routing out other factors.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
-
+ Thanks to this experiment we knew, that our pegRNAs work not only in HEK, but also in epithelial cells that express CFTR F508del.
</p>
</p>
</div>
<div className="box" >
<p id="peg4">
<H3 text="peg4" id="peg4head"/>
+ <p>
+ In this fourth iteration, we aimed to transfer our findings in optimizing the pegRNAs, generated in previous iterations, to the genomic CFTR context. To this end we modified our pegRNAs to be used in the CFTR gene editing process.
+ </p>
<H4 text="Design" id="design-head"/>
<p>
-
+ Using the pegFinder software and our acquired expertise in creating pegRNAs, we designed the new variants specifically tailored to the genomic CFTR region. These pegRNAs included the same combinations of PBS and RTT lengths as the ones we created for our reporter plasmid. Notably, scaffold, spacer, PBS and a part of the RTT did not have to be changed from the reporter targeting to genome targeting pegRNAs. Of the created pegRNAs, we wanted to focus on testing the most effective four variants found in the previous cycles and also a variant designated comparatively ineffective to test for consistency of our reporter system.
</p>
<H4 text="Build" id="build-head"/>
<p>
-
+ The newly designed pegRNAs were ordered as separate components, identical to the process used for the pegRNAs targeting the reporter system. Each RNA had both a constant and variable region, which we assembled using Golden Gate cloning. Afterwards we confirmed the correctness and completeness of the cloning into the pU6-peg-GG-acceptor plasmid through colony PCR screening. Unfortunately to this point, we were not able to produce positive clones.
</p>
<H4 text="Test" id="test-head"/>
<p>
-
+ The next step is to test the correction of CFTR F508del using these pegRNAs in the CFBE41o- epithelial cells. Additionally, we also want to test the pegRNAs in primary cells derived from friend of the team and cystic fibrosis patient Max[link], testing whether our approaches are applicable not only in model systems, but also work in patient cells. To validate the editing efficiency of our designed pegRNAs were going to co-transfect a plasmid carrying an eYFP variant which is sensitive to chloride and iodide ion concentrations<TabScrollLink tab="tab-pegrna" num="3" scrollId="desc-3"/><TabScrollLink tab="tab-pegrna" num="4" scrollId="desc-4"/>. The intensity of the fluorescence correlates with these ion concentrations, which in turn reflects the functionality of the CFTR channel. This enables us to evaluate the editing efficiency of the different pegRNA variants on a phenotypic level. After 72 hours, we are going to perform a final analysis using FACS to quantify the results and determine the editing efficiency of each pegRNA. Secondly, we wanted to detect the editing on a genomic level by facilitating a qPCR with a primer specific only to the corrected F508del locus.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
-
+ With this experiment we hope to achieve two things: Firstly, we want to examine whether optimizations of pegRNAs designed for our reporter system actually transfer to the genomic CFTR targeting. Secondly and most importantly, we want to find out whether we actually created an effective gene editing strategy for the genomic context of CFTR, thereby providing a foundation for a future gene therapy with high efficiency and precision when used with the right prime editor.
</p>
</p>
</div>
<div className="box" >
<p id="peg5">
<H3 text="peg5" id="peg5head"/>
+ <p>
+ In this final iteration, we focus on the outlook for future modifications and optimizations of our pegRNA design. These concepts are meant to further improve both the stability and editing efficiency through additional research and the implementation of new design strategies.
+ </p>
<H4 text="Design" id="design-head"/>
<p>
-
+ As we continued to refine our approach, further literature research was conducted, and new design ideas considered. The overarching goal remained to enhance both the stability and editing efficiency of the pegRNAs. One concept we are already exploring involves the incorporation of 3’ and 5’ UTRs (Untranslated Regions)<TabScrollLink tab="tab-pegrna" num="5" scrollId="desc-5"/>. These elements, typically found in mRNA, could be added to the pegRNA to increase its stability.
+ </p>
+ <p>
+ Another promising idea is the use of circular RNA (circRNA)<TabScrollLink tab="tab-pegrna" num="6" scrollId="desc-6"/>, which could provide additional stability by maintaining the closed-loop structure of the pegRNA. This would prevent degradation and increase the longevity of the pegRNA in the cell.
+ </p>
+ <p>
+ Additionally, further nucleotide modifications could be explored, such as experimenting with alternative silent edits to see if this leads to improved editing efficiency. We also nucleotide substitutions in the scaffold region to enhance RNA-binding affinity to the protein complex could be of use.
</p>
<H4 text="Build" id="build-head"/>
<p>
-
+ To implement these new design features, the individual components, such as UTRs, would need to be cloned into the existing pegRNAs. If we pursue alternative silent edits, the pegRNA sequences would need to be redesigned, ordered, and re-cloned. The circular RNA would also require a new assembly method to achieve the desired structure. However, the fundamental workflow would remain consistent with the processes used in previous iterations.
</p>
<H4 text="Test" id="test-head"/>
<p>
-
+ To maintain consistency and comparability, the same testing protocols used for the previous pegRNA screening would be applied. This includes co-transfection in the appropriate cell lines, fluorescence-based readouts for editing efficiency, and FACS analysis. By keeping the experimental conditions the same, we can ensure that the effects of the new modifications can be accurately assessed and compared to previous results.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
-
+ From these tests, we would aim to derive new insights not only specific to our particular context but also for pegRNA design as a whole. These future modifications could also yield valuable information on how to further improve the overall efficiency and stability of pegRNAs, contributing to the broader field of gene editing.
</p>
</p>
</div>
diff --git a/src/sources/eng-peg-sources.tsx b/src/sources/eng-peg-sources.tsx
index d429a543a8e1229ad513ea418f7fd5e341b81eba..81ec94d95483ae57b5bdd441fb09424d05c68cc0 100644
--- a/src/sources/eng-peg-sources.tsx
+++ b/src/sources/eng-peg-sources.tsx
@@ -10,5 +10,92 @@ export default function EngPegsources(){
const bibtexSources = [
-
+`
+ @article{Chow_Chen_Shen_Chen_2021,
+ title = {A web tool for the design of prime-editing guide RNAs},
+ author = {Chow, Ryan D. and Chen, Jennifer S. and Shen, Johanna and Chen, Sidi},
+ year = 2021,
+ month = feb,
+ journal = {Nature Biomedical Engineering},
+ publisher = {Nature Publishing Group},
+ volume = 5,
+ number = 2,
+ pages = {190–194},
+ doi = {10.1038/s41551-020-00622-8},
+ issn = {2157-846X},
+ rights = {2020 The Author(s), under exclusive licence to Springer Nature Limited},
+ abstractnote = {Prime editing enables diverse genomic alterations to be written into target sites without requiring double-strand breaks or donor templates. The design of prime-editing guide RNAs (pegRNAs), which must be customized for each edit, can however be complex and time consuming. Compared with single guide RNAs (sgRNAs), pegRNAs have an additional 3′ extension composed of a primer binding site and a reverse-transcription template. Here we report a web tool, which we named pegFinder (http://pegfinder.sidichenlab.org), for the rapid design of pegRNAs from reference and edited DNA sequences. pegFinder can incorporate sgRNA on-target and off-target scoring predictions into its ranking system, and nominates secondary nicking sgRNAs for increasing editing efficiency. CRISPR-associated protein 9 variants with expanded targeting ranges are also supported. To facilitate downstream experimentation, pegFinder produces a comprehensive table of candidate pegRNAs, along with oligonucleotide sequences for cloning.},
+ language = {en}
+ }
+`,`
+ @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}
+ }
+`,`
+ @article{Galietta_Haggie_Verkman_2001,
+ title = {Green fluorescent protein-based halide indicators with improved chloride and iodide affinities},
+ author = {Galietta, Luis J.V and Haggie, Peter M and Verkman, A.s},
+ year = 2001,
+ journal = {FEBS Letters},
+ volume = 499,
+ number = 3,
+ pages = {220–224},
+ doi = {10.1016/S0014-5793(01)02561-3},
+ issn = {1873-3468},
+ rights = {FEBS Letters 499 (2001) 1873-3468 © 2015 Federation of European Biochemical Societies},
+ abstractnote = {The green fluorescent protein YFP-H148Q is sensitive to halides by a mechanism involving halide binding and a shift in pK a. However, a limitation of YFP-H148Q is its low halide sensitivity, with K d>100 mM for Cl−. Indicators with improved sensitivities are needed for cell transport studies, particularly in drug discovery by high-throughput screening, and for measurement of Cl− concentration in subcellular organelles. YFP-H148Q libraries were generated in which pairs of residues in the vicinity of the halide binding site were randomly mutated. An automated procedure was developed to screen bacterial colonies for improved halide sensitivity. Analysis of 1536 clones revealed improved anion sensitivities with K d down to 2 mM for I− (I152L), 40 mM for Cl− (V163S), and 10 mM for NO3 − (I152L). The anion-sensitive mechanism of these indicators was established and their utility in cells was demonstrated using transfected cells expressing the cystic fibrosis transmembrane conductance regulator chloride channel.},
+ language = {en}
+ }
+`,`
+ @article{Bulcaen_Kortleven_Liu_Maule_Dreano_Kelly_Ensinck_Thierie_Smits_Ciciani_2024,
+ title = {Prime editing functionally corrects cystic fibrosis-causing CFTR mutations in human organoids and airway epithelial cells},
+ author = {Bulcaen, Mattijs and Kortleven, Phéline and Liu, Ronald B. and Maule, Giulia and Dreano, Elise and Kelly, Mairead and Ensinck, Marjolein M. and Thierie, Sam and Smits, Maxime and Ciciani, Matteo and Hatton, Aurelie and Chevalier, Benoit and Ramalho, Anabela S. and Casadevall i Solvas, Xavier and Debyser, Zeger and Vermeulen, François and Gijsbers, Rik and Sermet-Gaudelus, Isabelle and Cereseto, Anna and Carlon, Marianne S.},
+ year = 2024,
+ month = may,
+ journal = {Cell Reports Medicine},
+ pages = 101544,
+ doi = {10.1016/j.xcrm.2024.101544},
+ issn = {2666-3791},
+ abstractnote = {Prime editing is a recent, CRISPR-derived genome editing technology capable of introducing precise nucleotide substitutions, insertions, and deletions. Here, we present prime editing approaches to correct L227R- and N1303K-CFTR, two mutations that cause cystic fibrosis and are not eligible for current market-approved modulator therapies. We show that, upon DNA correction of the CFTR gene, the complex glycosylation, localization, and, most importantly, function of the CFTR protein are restored in HEK293T and 16HBE cell lines. These findings were subsequently validated in patient-derived rectal organoids and human nasal epithelial cells. Through analysis of predicted and experimentally identified candidate off-target sites in primary stem cells, we confirm previous reports on the high prime editor (PE) specificity and its potential for a curative CF gene editing therapy. To facilitate future screening of genetic strategies in a translational CF model, a machine learning algorithm was developed for dynamic quantification of CFTR function in organoids (DETECTOR: “detection of targeted editing of CFTR in organoidsâ€).}
+ }
+`,`
+ @article{Renz_Valdivia-Francia_Sendoel_2020,
+ title = {Some like it translated: small ORFs in the 5′UTR},
+ author = {Renz, Peter F. and Valdivia-Francia, Fabiola and Sendoel, Ataman},
+ year = 2020,
+ month = nov,
+ journal = {Experimental Cell Research},
+ volume = 396,
+ number = 1,
+ pages = 112229,
+ doi = {10.1016/j.yexcr.2020.112229},
+ issn = {0014-4827},
+ abstractnote = {The 5′ untranslated region (5′UTR) is critical in determining post-transcriptional control, which is partly mediated by short upstream open reading frames (uORFs) present in half of mammalian transcripts. uORFs are generally considered to provide functionally important repression of the main-ORF by engaging initiating ribosomes, but under specific environmental conditions such as cellular stress, uORFs can become essential to activate the translation of the main coding sequence. In addition, a growing number of uORF-encoded bioactive microproteins have been described, which have the potential to significantly increase cellular protein diversity. Here we review the diverse cellular contexts in which uORFs play a critical role and discuss the molecular mechanisms underlying their function and regulation. The progress over the last decades in dissecting uORF function suggests that the 5′UTR remains an exciting frontier towards understanding how the cellular proteome is shaped in health and disease.}
+ }
+`,`
+ @article{Liang_He_Zhao_Zhu_Hu_Liu_Gao_Liu_Zhang_Qiu_2024,
+ title = {Prime editing using CRISPR-Cas12a and circular RNAs in human cells},
+ author = {Liang, Ronghong and He, Zixin and Zhao, Kevin Tianmeng and Zhu, Haocheng and Hu, Jiacheng and Liu, Guanwen and Gao, Qiang and Liu, Meiyan and Zhang, Rui and Qiu, Jin-Long and Gao, Caixia},
+ year = 2024,
+ month = jan,
+ journal = {Nature Biotechnology},
+ doi = {10.1038/s41587-023-02095-x},
+ issn = {1087-0156, 1546-1696},
+ url = {https://www.nature.com/articles/s41587-023-02095-x},
+ language = {en}
+ }
+`
]
\ No newline at end of file