Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases.
Journal
Nature biotechnology
ISSN: 1546-1696
Titre abrégé: Nat Biotechnol
Pays: United States
ID NLM: 9604648
Informations de publication
Date de publication:
04 2023
04 2023
Historique:
received:
30
04
2022
accepted:
23
09
2022
medline:
19
4
2023
pubmed:
25
11
2022
entrez:
24
11
2022
Statut:
ppublish
Résumé
Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We demonstrate integration of sequences as large as ~36 kilobases at multiple genomic loci across three human cell lines, primary T cells and non-dividing primary human hepatocytes. To augment PASTE, we discovered 25,614 serine integrases and cognate attachment sites from metagenomes and engineered orthologs with higher activity and shorter recognition sequences for efficient programmable integration. PASTE has editing efficiencies similar to or exceeding those of homology-directed repair and non-homologous end joining-based methods, with activity in non-dividing cells and in vivo with fewer detectable off-target events. PASTE expands the capabilities of genome editing by allowing large, multiplexed gene insertion without reliance on DNA repair pathways.
Identifiants
pubmed: 36424489
doi: 10.1038/s41587-022-01527-4
pii: 10.1038/s41587-022-01527-4
pmc: PMC10257351
mid: NIHMS1902911
doi:
Substances chimiques
Integrases
EC 2.7.7.-
DNA
9007-49-2
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, N.I.H., Intramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
500-512Subventions
Organisme : NHLBI NIH HHS
ID : UH3 HL147367
Pays : United States
Organisme : NHLBI NIH HHS
ID : UG3 HL147367
Pays : United States
Organisme : NIBIB NIH HHS
ID : R01 EB031957
Pays : United States
Organisme : NHGRI NIH HHS
ID : R56 HG011857
Pays : United States
Organisme : NIAID NIH HHS
ID : R21 AI149694
Pays : United States
Commentaires et corrections
Type : CommentIn
Type : CommentIn
Type : CommentIn
Informations de copyright
© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.
Références
Elife. 2021 Oct 06;10:
pubmed: 34612202
Nat Med. 2019 Feb;25(2):229-233
pubmed: 30664785
Cell. 2016 Jan 14;164(1-2):29-44
pubmed: 26771484
Nat Biotechnol. 2022 May;40(5):731-740
pubmed: 34887556
Nat Rev Genet. 2018 Dec;19(12):770-788
pubmed: 30323312
Proc Natl Acad Sci U S A. 2003 Jul 22;100(15):8688-91
pubmed: 12837939
Nature. 2019 Jul;571(7764):219-225
pubmed: 31189177
Curr Gene Ther. 2013 Dec;13(6):421-33
pubmed: 24279313
Lancet Respir Med. 2016 Aug;4(8):e37-e38
pubmed: 27377414
Cold Spring Harb Protoc. 2019 May 1;2019(5):
pubmed: 31043559
Nucleic Acids Res. 2012 Nov;40(21):11163-72
pubmed: 23019222
PeerJ. 2015 May 28;3:e985
pubmed: 26038737
Nat Biotechnol. 2022 Feb;40(2):218-226
pubmed: 34650269
Mol Genet Genomics. 2006 Aug;276(2):135-46
pubmed: 16699779
Nucleic Acids Res. 2019 Sep 5;47(15):8126-8135
pubmed: 31429873
Nucleic Acids Res. 2016 Nov 16;44(20):9758-9770
pubmed: 27515511
Nucleic Acids Res. 2015 Jan;43(Database issue):D789-98
pubmed: 25428349
Curr Gene Ther. 2006 Dec;6(6):633-45
pubmed: 17168696
Mol Ther Methods Clin Dev. 2020 Jun 18;18:390-401
pubmed: 32695841
Nat Biotechnol. 2007 Aug;25(8):903-10
pubmed: 17664939
Nucleic Acids Res. 2011 Nov;39(21):9316-28
pubmed: 21849325
Nat Biotechnol. 2013 Sep;31(9):827-32
pubmed: 23873081
Nucleic Acids Res. 2020 Oct 9;48(18):10590-10601
pubmed: 32986839
Elife. 2019 Nov 01;8:
pubmed: 31674908
Nat Commun. 2022 Jul 20;13(1):4152
pubmed: 35858965
Nucleic Acids Res. 2015 Apr 20;43(7):e47
pubmed: 25605792
Mol Cell. 2012 Aug 24;47(4):497-510
pubmed: 22920291
Gene Ther. 2006 Aug;13(15):1188-90
pubmed: 16672982
Nature. 2017 Mar 2;543(7643):113-117
pubmed: 28225754
Science. 2019 Jul 5;365(6448):48-53
pubmed: 31171706
Nat Biotechnol. 2020 Jul;38(7):824-844
pubmed: 32572269
J Mol Biol. 2007 Mar 30;367(3):802-13
pubmed: 17289078
Cell. 1997 Nov 14;91(4):501-10
pubmed: 9390559
Bioinformatics. 2020 Apr 1;36(7):2272-2274
pubmed: 31821414
Hum Mutat. 2013 Nov;34(11):1537-1546
pubmed: 23946133
PLoS Genet. 2013 May;9(5):e1003490
pubmed: 23658531
Nat Biotechnol. 2022 Feb;40(2):227-234
pubmed: 34650270
Nat Commun. 2018 Jul 6;9(1):2629
pubmed: 29980667
Nat Methods. 2022 Mar;19(3):331-340
pubmed: 35228726
J Virol. 2005 Dec;79(23):14793-803
pubmed: 16282479
Nat Biotechnol. 2010 Dec;28(12):1287-9
pubmed: 21102455
Nature. 2019 Dec;576(7785):149-157
pubmed: 31634902
BMC Biotechnol. 2013 Oct 20;13:87
pubmed: 24139482
FEBS Lett. 2020 Dec;594(24):4338-4356
pubmed: 32970841
Mol Cell Biol. 1994 Dec;14(12):8096-106
pubmed: 7969147
Bioinformatics. 2010 Jan 1;26(1):139-40
pubmed: 19910308
Nature. 2017 Nov 23;551(7681):464-471
pubmed: 29160308
Genome Biol. 2014 Feb 03;15(2):R29
pubmed: 24485249
PLoS Comput Biol. 2011 Oct;7(10):e1002195
pubmed: 22039361
Proc Natl Acad Sci U S A. 2009 Mar 31;106(13):5053-8
pubmed: 19282480
Nucleic Acids Res. 2020 Sep 18;48(16):9067-9081
pubmed: 32687165
Trends Biotechnol. 2018 Aug;36(8):770-786
pubmed: 29685818
Cold Spring Harb Protoc. 2019 May 1;2019(5):
pubmed: 31043560
Bioinformatics. 2013 Jan 1;29(1):15-21
pubmed: 23104886
Nucleic Acids Res. 2011 Sep 1;39(17):7868-78
pubmed: 21653554
Nature. 2016 Apr 20;533(7603):420-4
pubmed: 27096365
Bioinformatics. 2009 Mar 15;25(6):765-71
pubmed: 19176553
Cell. 2014 Jun 5;157(6):1262-1278
pubmed: 24906146
Genome Biol. 2015 Dec 15;16:280
pubmed: 26671237
Synth Biol (Oxf). 2019;4(1):ysz018
pubmed: 31355344
Nucleic Acids Res. 2021 Jan 8;49(D1):D480-D489
pubmed: 33237286
BMC Bioinformatics. 2010 Mar 08;11:119
pubmed: 20211023
Cell Host Microbe. 2020 Nov 11;28(5):767
pubmed: 33181077
ACS Chem Biol. 2018 Feb 16;13(2):467-474
pubmed: 28892606
Science. 2013 Feb 15;339(6121):823-6
pubmed: 23287722
Hum Gene Ther. 2006 Nov;17(11):1077-94
pubmed: 17069535
Stat Appl Genet Mol Biol. 2004;3:Article3
pubmed: 16646809
Nucleic Acids Res. 2008 Jul 1;36(Web Server issue):W5-9
pubmed: 18440982
Science. 2013 Feb 15;339(6121):819-23
pubmed: 23287718
Methods. 2011 Apr;53(4):372-9
pubmed: 21195181
Elife. 2020 Mar 06;9:
pubmed: 32142408
Cold Spring Harb Protoc. 2020 Aug 3;2020(8):095513
pubmed: 32457039
ACS Synth Biol. 2019 Jan 18;8(1):16-24
pubmed: 30609349
Elife. 2014 Dec 15;3:e04766
pubmed: 25497837
Mol Cell. 2003 Nov;12(5):1101-11
pubmed: 14636570
CRISPR J. 2019 Dec;2(6):376-394
pubmed: 31742433
Nucleic Acids Res. 2015 Sep 30;43(17):e112
pubmed: 26007658
Nature. 2016 Dec 1;540(7631):144-149
pubmed: 27851729