rights={2019 The Author(s), under exclusive licence to Springer Nature Limited}, ISSN={1476-4687},
year={2008},
DOI={10.1038/s41586-019-1711-4},
month={ },
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.},
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, pages={149–157}, language={en} }
volume={24},
@article{Doman_Pandey_Neugebauer_An_Davis_Randolph_McElroy_Gao_Raguram_Richter_etal._2023,title={Phage-assistedevolutionandproteinengineeringyieldcompact,efficientprimeeditors}, volume={186}, ISSN={0092-8674, 1097-4172}, DOI={10.1016/j.cell.2023.07.039}, number={18}, journal={Cell}, publisher={Elsevier}, 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}, month=aug, pages={3983-4002.e26}, language={English} }
journal={Cadernos de Saúde Pública},
@article{Jinek_Chylinski_Fonfara_Hauer_Doudna_Charpentier_2012,title={A programmable dual RNA-guided DNA endonuclease in adaptive bacterial immunity},volume={337},ISSN={0036-8075},DOI={10.1126/science.1225829},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.},number={6096},journal={Science (New York, N.Y.)},author={Jinek, Martin and Chylinski, Krzysztof and Fonfara, Ines and Hauer, Michael and Doudna, Jennifer A. and Charpentier, Emmanuelle},year={2012},month=aug,pages={816–821}}
doi={10.1590/S0102-311X2008001600002}
@article{Nelson_Randolph_Shen_Everette_Chen_Anzalone_An_Newby_Chen_Hsu_etal._2022,title={EngineeredpegRNAsimproveprimeeditingefficiency}, volume={40}, rights={2021 The Author(s), under exclusive licence to Springer Nature America, Inc.}, ISSN={1546-1696}, DOI={10.1038/s41587-021-01039-7}, abstractNote={Prime editing enables the installation of virtually any combination of point mutations, small insertions or small deletions in the DNA of living cells. A prime editing guide RNA (pegRNA) directs the prime editor protein to the targeted locus and also encodes the desired edit. Here we show that degradation of the 3′ region of the pegRNA that contains the reverse transcriptase template and the primer binding site can poison the activity of prime editing systems, impeding editing efficiency. We incorporated structured RNA motifs to the 3′ terminus of pegRNAs that enhance their stability and prevent degradation of the 3′ extension. The resulting engineered pegRNAs (epegRNAs) improve prime editing efficiency 3–4-fold in HeLa, U2OS and K562 cells and in primary human fibroblasts without increasing off-target editing activity. We optimized the choice of 3′ structural motif and developed pegLIT, a computational tool to identify non-interfering nucleotide linkers between pegRNAs and 3′ motifs. Finally, we showed that epegRNAs enhance the efficiency of the installation or correction of disease-relevant mutations.}, number={3}, journal={Nature Biotechnology}, publisher={Nature Publishing Group}, author={Nelson, James W. and Randolph, Peyton B. and Shen, Simon P. and Everette, Kelcee A. and Chen, Peter J. and Anzalone, Andrew V. and An, Meirui and Newby, Gregory A. and Chen, Jonathan C. and Hsu, Alvin and Liu, David R.}, year={2022}, month=mar, pages={402–410}, language={en} }
}
@article{Sousa_Hemez_Lei_Traore_Kulhankova_Newby_Doman_Oye_Pandey_Karp_etal._2024,title={SystematicoptimizationofprimeeditingfortheefficientfunctionalcorrectionofCFTRF508delinhumanairwayepithelialcells}, rights={2024 The Author(s)}, ISSN={2157-846X}, DOI={10.1038/s41551-024-01233-3}, 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.}, journal={Nature Biomedical Engineering}, publisher={Nature Publishing Group}, volume={}, 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.}, year={2024}, month=jul, pages={1–15}, language={en} }
<span property="schema:name"> Systematic optimization of prime editing for the efficient functional correction of CFTR F508del in human airway epithelial cells. </span>
<span property="schema:name"> Unravelling the Regions of Mutant F508del-CFTR More Susceptible to the Action of Four Cystic Fibrosis Correctors. </span>
<i property="schema:publisher" typeof="schema:Organization"> International Journal of Molecular Sciences</i>
@@ -58,6 +58,18 @@ export function Description() {
<p>Text about CFTR <LoremMedium/></p>
</div>
</div>
<h3>ΔF508</h3>
<p>A multitude of mutations in the CFTR gene, exceeding 1,000, are responsible for the development of cystic
fibrosis. The most prevalent variant is F508del, observed in approximately 70% of affected individuals of
Caucasian descent in Canada, Northern Europe, and the United States <ahref="#desc-14"><sup>14</sup></a>. It is estimated that around 90% of
the European population and people of European heritage with cystic fibrosis carry at least one F508del
variant <ahref="#desc-15"><sup>15,</sup></a><ahref="#desc-16"><sup>16</sup></a>. Analyses have demonstrated that the F508del mutation originated in Western Europe at least
5,000 years ago <ahref="#desc-15"><sup>15</sup></a>. </p>
<p>It is a deletion of the three nucleotides "CTT" at position 508, which removes the phenylalanine residue
without causing a frameshift. This deletion leads to defects in the kinetic and thermodynamic folding
of the NBD1 domain <ahref="#desc-16"><sup>16</sup></a>. However, this not only leads to misfolding of CFTR but also to defects in
trafficking and premature degradation, resulting in reduced surface expression of CFTR <ahref="#desc-17"><sup>17</sup></a>. </p>
@@ -90,8 +102,7 @@ export function Description() {
<p>We chose to focus on CF and specifically the ΔF508 mutation due to its prevalence and the severe impact it has on patients' lives. Additionally, our team includes members who have close friends affected by this condition, giving us a personal connection and a strong motivation to find a solution. By targeting the ΔF508 mutation, we aim to develop a therapy that could potentially, not only benefit many CF patients and make a significant improvement in their lives, but also can serve as a template, which research groups can use to target other genetic diseases. </p>
<spanproperty="schema:name"> Unravelling the Regions of Mutant F508del-CFTR More Susceptible to the Action of Four Cystic Fibrosis Correctors. </span>
<iproperty="schema:publisher"typeof="schema:Organization"> International Journal of Molecular Sciences</i>
