Optimized nickase- and nuclease-based prime editing in human and mouse cells.
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
11 10 2021
11 10 2021
Historique:
accepted:
16
09
2021
revised:
26
08
2021
received:
21
07
2021
pubmed:
18
9
2021
medline:
18
12
2021
entrez:
17
9
2021
Statut:
ppublish
Résumé
Precise genomic modification using prime editing (PE) holds enormous potential for research and clinical applications. In this study, we generated all-in-one prime editing (PEA1) constructs that carry all the components required for PE, along with a selection marker. We tested these constructs (with selection) in HEK293T, K562, HeLa and mouse embryonic stem (ES) cells. We discovered that PE efficiency in HEK293T cells was much higher than previously observed, reaching up to 95% (mean 67%). The efficiency in K562 and HeLa cells, however, remained low. To improve PE efficiency in K562 and HeLa, we generated a nuclease prime editor and tested this system in these cell lines as well as mouse ES cells. PE-nuclease greatly increased prime editing initiation, however, installation of the intended edits was often accompanied by extra insertions derived from the repair template. Finally, we show that zygotic injection of the nuclease prime editor can generate correct modifications in mouse fetuses with up to 100% efficiency.
Identifiants
pubmed: 34534334
pii: 6371974
doi: 10.1093/nar/gkab792
pmc: PMC8501948
doi:
Substances chimiques
CRISPR-Associated Protein 9
EC 3.1.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
10785-10795Informations de copyright
© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.
Références
PLoS One. 2017 Dec 6;12(12):e0187236
pubmed: 29211736
Nat Commun. 2020 Oct 23;11(1):5352
pubmed: 33097693
Nature. 2016 Jan 28;529(7587):490-5
pubmed: 26735016
Cell. 2013 May 9;153(4):910-8
pubmed: 23643243
Nat Biotechnol. 2013 Sep;31(9):827-32
pubmed: 23873081
Nucleic Acids Res. 2021 Jul 2;49(W1):W499-W504
pubmed: 33939828
Plant Biotechnol J. 2021 Mar;19(3):415-417
pubmed: 33091225
Genome Biol. 2021 Mar 16;22(1):83
pubmed: 33722289
Nature. 2019 Dec;576(7785):149-157
pubmed: 31634902
Nat Biotechnol. 2021 Aug;39(8):923-927
pubmed: 33767395
Cell. 2013 Sep 12;154(6):1370-9
pubmed: 23992847
Nat Biotechnol. 2020 May;38(5):582-585
pubmed: 32393904
Science. 2016 Jan 1;351(6268):84-8
pubmed: 26628643
Nature. 2018 Aug;560(7717):E8-E9
pubmed: 30089922
Plant Commun. 2020 Apr 08;1(3):100043
pubmed: 33367239
Cell. 2013 Sep 12;154(6):1380-9
pubmed: 23992846
Nature. 2018 Sep;561(7723):416-419
pubmed: 30209390
Genome Biol. 2021 Jun 3;22(1):170
pubmed: 34082781
Genes (Basel). 2020 May 06;11(5):
pubmed: 32384610
CRISPR J. 2021 Feb;4(1):120-131
pubmed: 33571043
Nat Commun. 2021 Apr 9;12(1):2121
pubmed: 33837189
Proc Natl Acad Sci U S A. 2021 Jan 5;118(1):
pubmed: 33443210
Nat Biotechnol. 2021 Apr 29;:
pubmed: 33927418
Genome Biol. 2020 Oct 6;21(1):257
pubmed: 33023639
Nat Biotechnol. 2013 Sep;31(9):822-6
pubmed: 23792628
Sci Adv. 2021 Apr 30;7(18):
pubmed: 33931459
Mol Plant. 2020 May 4;13(5):671-674
pubmed: 32222486
Cell Discov. 2020 Apr 28;6:27
pubmed: 32351707
Nat Protoc. 2013 Nov;8(11):2281-2308
pubmed: 24157548