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Chromatin structure and context-dependent sequence features control prime editing efficiency

Prime editing (PE) is a highly versatile CRISPR–Cas9 genome editing technique. The current constructs, however, have variable efficiency and may require laborious experimental optimization. This study presents statistical models for learning the salient epigenomic and sequence features of target sit...

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Autores principales: Kim, Somang, Yuan, Jimmy B., Woods, Wendy S., Newton, Destry A., Perez-Pinera, Pablo, Song, Jun S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10344898/
https://www.ncbi.nlm.nih.gov/pubmed/37456665
http://dx.doi.org/10.3389/fgene.2023.1222112
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author Kim, Somang
Yuan, Jimmy B.
Woods, Wendy S.
Newton, Destry A.
Perez-Pinera, Pablo
Song, Jun S.
author_facet Kim, Somang
Yuan, Jimmy B.
Woods, Wendy S.
Newton, Destry A.
Perez-Pinera, Pablo
Song, Jun S.
author_sort Kim, Somang
collection PubMed
description Prime editing (PE) is a highly versatile CRISPR–Cas9 genome editing technique. The current constructs, however, have variable efficiency and may require laborious experimental optimization. This study presents statistical models for learning the salient epigenomic and sequence features of target sites modulating the editing efficiency and provides guidelines for designing optimal PEs. We found that both regional constitutive heterochromatin and local nucleosome occlusion of target sites impede editing, while position-specific G/C nucleotides in the primer-binding site (PBS) and reverse transcription (RT) template regions of PE guide RNA (pegRNA) yield high editing efficiency, especially for short PBS designs. The presence of G/C nucleotides was most critical immediately 5’ to the protospacer adjacent motif (PAM) site for all designs. The effects of different last templated nucleotides were quantified and observed to depend on the length of both PBS and RT templates. Our models found AGG to be the preferred PAM and detected a guanine nucleotide four bases downstream of the PAM to facilitate editing, suggesting a hitherto-unrecognized interaction with Cas9. A neural network interpretation method based on nonextensive statistical mechanics further revealed multi-nucleotide preferences, indicating dependency among several bases across pegRNA. Our work clarifies previous conflicting observations and uncovers context-dependent features important for optimizing PE designs.
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spelling pubmed-103448982023-07-15 Chromatin structure and context-dependent sequence features control prime editing efficiency Kim, Somang Yuan, Jimmy B. Woods, Wendy S. Newton, Destry A. Perez-Pinera, Pablo Song, Jun S. Front Genet Genetics Prime editing (PE) is a highly versatile CRISPR–Cas9 genome editing technique. The current constructs, however, have variable efficiency and may require laborious experimental optimization. This study presents statistical models for learning the salient epigenomic and sequence features of target sites modulating the editing efficiency and provides guidelines for designing optimal PEs. We found that both regional constitutive heterochromatin and local nucleosome occlusion of target sites impede editing, while position-specific G/C nucleotides in the primer-binding site (PBS) and reverse transcription (RT) template regions of PE guide RNA (pegRNA) yield high editing efficiency, especially for short PBS designs. The presence of G/C nucleotides was most critical immediately 5’ to the protospacer adjacent motif (PAM) site for all designs. The effects of different last templated nucleotides were quantified and observed to depend on the length of both PBS and RT templates. Our models found AGG to be the preferred PAM and detected a guanine nucleotide four bases downstream of the PAM to facilitate editing, suggesting a hitherto-unrecognized interaction with Cas9. A neural network interpretation method based on nonextensive statistical mechanics further revealed multi-nucleotide preferences, indicating dependency among several bases across pegRNA. Our work clarifies previous conflicting observations and uncovers context-dependent features important for optimizing PE designs. Frontiers Media S.A. 2023-06-29 /pmc/articles/PMC10344898/ /pubmed/37456665 http://dx.doi.org/10.3389/fgene.2023.1222112 Text en Copyright © 2023 Kim, Yuan, Woods, Newton, Perez-Pinera and Song. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Genetics
Kim, Somang
Yuan, Jimmy B.
Woods, Wendy S.
Newton, Destry A.
Perez-Pinera, Pablo
Song, Jun S.
Chromatin structure and context-dependent sequence features control prime editing efficiency
title Chromatin structure and context-dependent sequence features control prime editing efficiency
title_full Chromatin structure and context-dependent sequence features control prime editing efficiency
title_fullStr Chromatin structure and context-dependent sequence features control prime editing efficiency
title_full_unstemmed Chromatin structure and context-dependent sequence features control prime editing efficiency
title_short Chromatin structure and context-dependent sequence features control prime editing efficiency
title_sort chromatin structure and context-dependent sequence features control prime editing efficiency
topic Genetics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10344898/
https://www.ncbi.nlm.nih.gov/pubmed/37456665
http://dx.doi.org/10.3389/fgene.2023.1222112
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