Functional profiling of single CRISPR/Cas9-edited human long-term hematopoietic stem cells.
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
18 10 2019
18 10 2019
Historique:
received:
05
04
2019
accepted:
29
09
2019
entrez:
20
10
2019
pubmed:
20
10
2019
medline:
25
1
2020
Statut:
epublish
Résumé
In the human hematopoietic system, rare self-renewing multipotent long-term hematopoietic stem cells (LT-HSCs) are responsible for the lifelong production of mature blood cells and are the rational target for clinical regenerative therapies. However, the heterogeneity in the hematopoietic stem cell compartment and variable outcomes of CRISPR/Cas9 editing make functional interrogation of rare LT-HSCs challenging. Here, we report high efficiency LT-HSC editing at single-cell resolution using electroporation of modified synthetic gRNAs and Cas9 protein. Targeted short isoform expression of the GATA1 transcription factor elicit distinct differentiation and proliferation effects in single highly purified LT-HSC when analyzed with functional in vitro differentiation and long-term repopulation xenotransplantation assays. Our method represents a blueprint for systematic genetic analysis of complex tissue hierarchies at single-cell resolution.
Identifiants
pubmed: 31628330
doi: 10.1038/s41467-019-12726-0
pii: 10.1038/s41467-019-12726-0
pmc: PMC6802205
doi:
Substances chimiques
GATA1 Transcription Factor
0
GATA1 protein, human
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
4730Subventions
Organisme : NIAID NIH HHS
ID : R01 AI132963
Pays : United States
Organisme : NIDDK NIH HHS
ID : U01 DK104218
Pays : United States
Organisme : NIDDK NIH HHS
ID : UC4 DK104218
Pays : United States
Références
Nucleic Acids Res. 2014 Dec 16;42(22):e168
pubmed: 25300484
Cell Rep. 2016 Oct 25;17(5):1453-1461
pubmed: 27783956
Blood. 2003 Aug 1;102(3):981-6
pubmed: 12649131
Blood. 2003 Jun 1;101(11):4301-4
pubmed: 12586620
EMBO J. 2001 Sep 17;20(18):5250-60
pubmed: 11566888
Nucleic Acids Res. 2018 Jul 6;46(12):e71
pubmed: 29584876
Curr Opin Hematol. 2017 Nov;24(6):481-488
pubmed: 28806273
Blood. 2014 May 15;123(20):3080-8
pubmed: 24652993
Science. 2005 Sep 23;309(5743):2033-7
pubmed: 16179473
Cell Stem Cell. 2014 Nov 6;15(5):643-52
pubmed: 25517468
Pediatr Blood Cancer. 2018 Jan;65(1):
pubmed: 28801981
PLoS One. 2018 Dec 12;13(12):e0209034
pubmed: 30540841
Blood. 2008 Jan 15;111(2):767-75
pubmed: 17901249
Elife. 2017 Sep 28;6:
pubmed: 28956530
J Immunol Methods. 2009 Aug 15;347(1-2):70-8
pubmed: 19567251
Nat Med. 2014 Jul;20(7):748-53
pubmed: 24952648
Blood. 2015 Aug 20;126(8):943-9
pubmed: 26186939
Nat Genet. 1999 Feb;21(2):169-75
pubmed: 9988267
Nat Genet. 2005 Jun;37(6):613-9
pubmed: 15895080
Science. 2011 Jul 8;333(6039):218-21
pubmed: 21737740
Cell Stem Cell. 2019 Apr 4;24(4):551-565.e8
pubmed: 30905619
Blood Adv. 2018 Apr 24;2(8):832-845
pubmed: 29650777
Nature. 2017 Jul 6;547(7661):104-108
pubmed: 28658204
Nat Med. 2018 Aug;24(8):1216-1224
pubmed: 30082871
Bioinformatics. 2014 May 15;30(10):1473-5
pubmed: 24463181
Curr Hematol Malig Rep. 2016 Oct;11(5):333-41
pubmed: 27510823
Stem Cell Reports. 2016 Oct 11;7(4):591-601
pubmed: 27618723
N Engl J Med. 2019 Mar 7;380(10):947-959
pubmed: 30855744
Blood. 2007 Sep 15;110(6):1933-41
pubmed: 17505015
Nature. 2016 Nov 17;539(7629):384-389
pubmed: 27820943
Nat Protoc. 2018 Feb;13(2):358-376
pubmed: 29370156
Proc Natl Acad Sci U S A. 2014 Feb 18;111(7):2548-53
pubmed: 24550281
Exp Hematol. 1989 Feb;17(2):145-53
pubmed: 2783573
Cell Stem Cell. 2017 Oct 5;21(4):547-555.e8
pubmed: 28985529
Blood. 2008 Dec 15;112(13):4793-807
pubmed: 19064739
Blood. 1999 May 1;93(9):2867-75
pubmed: 10216081
Pediatr Blood Cancer. 2014 Jul;61(7):1319-21
pubmed: 24453067
Blood. 2013 Dec 5;122(24):3908-17
pubmed: 24021668
Cancer Cell. 2019 Aug 12;36(2):123-138.e10
pubmed: 31303423
Blood. 2004 Apr 1;103(7):2480-9
pubmed: 14656875
Nat Commun. 2018 Jul 6;9(1):2641
pubmed: 29980686
Cell Stem Cell. 2015 Mar 5;16(3):302-13
pubmed: 25704240
Nature. 2014 Feb 20;506(7488):328-33
pubmed: 24522528
J Clin Invest. 2012 Jul;122(7):2439-43
pubmed: 22706301
Nucleic Acids Res. 2018 Jan 25;46(2):792-803
pubmed: 29216382
Blood. 2003 Jun 1;101(11):4298-300
pubmed: 12560215
Nat Genet. 1992 May;1(2):92-8
pubmed: 1302015
Nat Med. 2003 Jul;9(7):959-63
pubmed: 12796774
J Vis Exp. 2018 Apr 10;(134):
pubmed: 29708546
Science. 2016 Jan 8;351(6269):aab2116
pubmed: 26541609
Nature. 1990 Mar 29;344(6265):444-7
pubmed: 2320112
Mol Ther. 2019 Jan 2;27(1):137-150
pubmed: 30424953
Nat Biotechnol. 2015 Sep;33(9):985-989
pubmed: 26121415
Blood. 2010 Apr 22;115(16):3415-6
pubmed: 20413665
Nat Med. 2019 May;25(5):776-783
pubmed: 30911135
Blood. 1993 Aug 15;82(4):1071-9
pubmed: 8353273
Nat Genet. 2002 Sep;32(1):148-52
pubmed: 12172547
Haematologica. 2018 Aug;103(8):e336-e340
pubmed: 29567780
Am J Hum Genet. 2018 Dec 6;103(6):930-947
pubmed: 30503522
Mol Cell. 2017 Jul 20;67(2):348-354.e4
pubmed: 28732207