A simplified transposon mutagenesis method to perform phenotypic forward genetic screens in cultured cells.
Journal
BMC genomics
ISSN: 1471-2164
Titre abrégé: BMC Genomics
Pays: England
ID NLM: 100965258
Informations de publication
Date de publication:
17 Jun 2019
17 Jun 2019
Historique:
received:
17
04
2019
accepted:
06
06
2019
entrez:
19
6
2019
pubmed:
19
6
2019
medline:
17
3
2020
Statut:
epublish
Résumé
The introduction of genome-wide shRNA and CRISPR libraries has facilitated cell-based screens to identify loss-of-function mutations associated with a phenotype of interest. Approaches to perform analogous gain-of-function screens are less common, although some reports have utilized arrayed viral expression libraries or the CRISPR activation system. However, a variety of technical and logistical challenges make these approaches difficult for many labs to execute. In addition, genome-wide shRNA or CRISPR libraries typically contain of hundreds of thousands of individual engineered elements, and the associated complexity creates issues with replication and reproducibility for these methods. Here we describe a simple, reproducible approach using the SB transposon system to perform phenotypic cell-based genetic screens. This approach employs only three plasmids to perform unbiased, whole-genome transposon mutagenesis. We also describe a ligation-mediated PCR method that can be used in conjunction with the included software tools to map raw sequence data, identify candidate genes associated with phenotypes of interest, and predict the impact of recurrent transposon insertions on candidate gene function. Finally, we demonstrate the high reproducibility of our approach by having three individuals perform independent replicates of a mutagenesis screen to identify drivers of vemurafenib resistance in cultured melanoma cells. Collectively, our work establishes a facile, adaptable method that can be performed by labs of any size to perform robust, genome-wide screens to identify genes that influence phenotypes of interest.
Sections du résumé
BACKGROUND
BACKGROUND
The introduction of genome-wide shRNA and CRISPR libraries has facilitated cell-based screens to identify loss-of-function mutations associated with a phenotype of interest. Approaches to perform analogous gain-of-function screens are less common, although some reports have utilized arrayed viral expression libraries or the CRISPR activation system. However, a variety of technical and logistical challenges make these approaches difficult for many labs to execute. In addition, genome-wide shRNA or CRISPR libraries typically contain of hundreds of thousands of individual engineered elements, and the associated complexity creates issues with replication and reproducibility for these methods.
RESULTS
RESULTS
Here we describe a simple, reproducible approach using the SB transposon system to perform phenotypic cell-based genetic screens. This approach employs only three plasmids to perform unbiased, whole-genome transposon mutagenesis. We also describe a ligation-mediated PCR method that can be used in conjunction with the included software tools to map raw sequence data, identify candidate genes associated with phenotypes of interest, and predict the impact of recurrent transposon insertions on candidate gene function. Finally, we demonstrate the high reproducibility of our approach by having three individuals perform independent replicates of a mutagenesis screen to identify drivers of vemurafenib resistance in cultured melanoma cells.
CONCLUSIONS
CONCLUSIONS
Collectively, our work establishes a facile, adaptable method that can be performed by labs of any size to perform robust, genome-wide screens to identify genes that influence phenotypes of interest.
Identifiants
pubmed: 31208320
doi: 10.1186/s12864-019-5888-6
pii: 10.1186/s12864-019-5888-6
pmc: PMC6580595
doi:
Substances chimiques
DNA Transposable Elements
0
Vemurafenib
207SMY3FQT
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
497Subventions
Organisme : NCI NIH HHS
ID : P30 CA086862
Pays : United States
Organisme : NIGMS NIH HHS
ID : R25 GM116686
Pays : United States
Organisme : NIGMS NIH HHS
ID : T32 GM007337
Pays : United States
Organisme : NIGMS NIH HHS
ID : T32 GM067795
Pays : United States
Références
Nat Rev Genet. 2002 Mar;3(3):176-88
pubmed: 11972155
Science. 2014 Jan 3;343(6166):84-87
pubmed: 24336571
Curr Opin Genet Dev. 2018 Apr;49:85-94
pubmed: 29587177
Nucleic Acids Res. 2017 Jan 4;45(D1):D679-D686
pubmed: 27789686
Cancer Res. 2017 Mar 15;77(6):1357-1368
pubmed: 28108518
Nat Genet. 2015 Feb;47(2):142-50
pubmed: 25559195
J Mol Biol. 2002 Oct 25;323(3):441-52
pubmed: 12381300
Hepatology. 2013 Jan;57(1):120-30
pubmed: 22899566
Nat Protoc. 2017 Apr;12(4):828-863
pubmed: 28333914
Int J Cancer. 2017 Feb 15;140(4):853-863
pubmed: 27790711
J Invest Dermatol. 2013 Jan;133(1):239-48
pubmed: 22832494
Science. 2014 Jan 3;343(6166):80-4
pubmed: 24336569
Nat Genet. 2015 May;47(5):486-95
pubmed: 25848750
Proc Natl Acad Sci U S A. 2011 Jan 25;108(4):1531-6
pubmed: 21205896
Mol Ther. 2003 Jul;8(1):108-17
pubmed: 12842434
Genesis. 2009 Jun;47(6):404-8
pubmed: 19391106
Cancer Res. 2009 Oct 15;69(20):8150-6
pubmed: 19808965
Cancer Res. 2016 Feb 15;76(4):773-86
pubmed: 26676752
Nature. 2012 Feb 15;482(7386):529-33
pubmed: 22343890
J Immunol. 2013 Apr 15;190(8):4393-9
pubmed: 23475219
Nature. 2015 Jan 29;517(7536):583-8
pubmed: 25494202
Proc Natl Acad Sci U S A. 2016 Jun 14;113(24):E3384-93
pubmed: 27247392
Nat Biotechnol. 2014 Mar;32(3):267-73
pubmed: 24535568
Hepatology. 2018 Mar;67(3):924-939
pubmed: 28961327
Biosci Rep. 2017 Jul 27;37(4):
pubmed: 28754805
PLoS One. 2012;7(11):e42666
pubmed: 23166581
Cancer Res. 2019 Oct 1;79(19):5074-5087
pubmed: 31416844
Blood. 2013 May 23;121(21):4355-8
pubmed: 23591791
Semin Cancer Biol. 2010 Aug;20(4):261-8
pubmed: 20478384
BMC Genomics. 2014 Dec 19;15:1150
pubmed: 25526783
Nature. 2005 Jul 14;436(7048):221-6
pubmed: 16015321
Proc Natl Acad Sci U S A. 2005 May 3;102(18):6425-30
pubmed: 15851657
Nucleic Acids Res. 2013 Jan;41(Database issue):D1021-6
pubmed: 23193271
Cancer Res. 2017 Dec 1;77(23):6576-6588
pubmed: 28993411
Pharmacol Ther. 2018 Nov;191:178-189
pubmed: 29953899
Nature. 2011 Nov 23;480(7377):387-90
pubmed: 22113612
PLoS One. 2011;6(9):e24668
pubmed: 21931803
Proc Natl Acad Sci U S A. 2007 Sep 4;104(36):14406-11
pubmed: 17720809
Proc Natl Acad Sci U S A. 2012 Apr 17;109(16):5934-41
pubmed: 22421440
Nature. 2005 Jul 14;436(7048):272-6
pubmed: 16015333
Nucleic Acids Res. 2012 Aug;40(14):6693-712
pubmed: 22523082
Sci Rep. 2017 Dec 18;7(1):17693
pubmed: 29255251
Proc Natl Acad Sci U S A. 2017 Jun 20;114(25):E4951-E4960
pubmed: 28584132
Cell. 2015 Dec 3;163(6):1515-26
pubmed: 26627737
Mol Cell Biol. 2005 Mar;25(6):2085-94
pubmed: 15743807
Nat Rev Genet. 2002 May;3(5):356-69
pubmed: 11988761
Nat Genet. 2014 Jan;46(1):24-32
pubmed: 24316982
Nat Methods. 2015 Apr;12(4):357-60
pubmed: 25751142
Nat Genet. 2009 Jun;41(6):753-61
pubmed: 19412179
Nat Biotechnol. 2016 Aug;34(8):845-51
pubmed: 27398792
Nat Rev Genet. 2001 Sep;2(9):659-68
pubmed: 11533715
Cell. 2018 Apr 19;173(3):649-664.e20
pubmed: 29677511
Cancer Discov. 2013 Mar;3(3):350-62
pubmed: 23288408
Blood. 2011 Oct 27;118(17):4646-56
pubmed: 21828136