A single-chain and fast-responding light-inducible Cre recombinase as a novel optogenetic switch.
Cre/Lox
DNA recombination
S. cerevisiae
biochemistry
chemical biology
genetics
genome editing
genomics
human
optogenetics
synthetic biology
Journal
eLife
ISSN: 2050-084X
Titre abrégé: Elife
Pays: England
ID NLM: 101579614
Informations de publication
Date de publication:
23 02 2021
23 02 2021
Historique:
received:
20
07
2020
accepted:
22
02
2021
pubmed:
24
2
2021
medline:
29
1
2022
entrez:
23
2
2021
Statut:
epublish
Résumé
Optogenetics enables genome manipulations with high spatiotemporal resolution, opening exciting possibilities for fundamental and applied biological research. Here, we report the development of LiCre, a novel light-inducible Cre recombinase. LiCre is made of a single flavin-containing protein comprising the AsLOV2 photoreceptor domain of In a biologist’s toolkit, the Cre protein holds a special place. Naturally found in certain viruses, this enzyme recognises and modifies specific genetic sequences, creating changes that switch on or off whatever gene is close by. Genetically engineering cells or organisms so that they carry Cre and its target sequences allows scientists to control the activation of a given gene, often in a single tissue or organ. However, this relies on the ability to activate the Cre protein ‘on demand’ once it is in the cells of interest. One way to do so is to split the enzyme into two pieces, which can then reassemble when exposed to blue light. Yet, this involves the challenging step of introducing both parts separately into a tissue. Instead, Duplus-Bottin et al. engineered LiCre, a new system where a large section of the Cre protein is fused to a light sensor used by oats to detect their environment. LiCre is off in the dark, but it starts to recognize and modify Cre target sequences when exposed to blue light. Duplus-Bottin et al. then assessed how LiCre compares to the two-part Cre system in baker's yeast and human kidney cells. This showed that the new protein is less ‘incorrectly’ active in the dark, and can switch on faster under blue light. The improved approach could give scientists a better tool to study the role of certain genes at precise locations and time points, but also help them to harness genetic sequences for industry or during gene therapy.
Autres résumés
Type: plain-language-summary
(eng)
In a biologist’s toolkit, the Cre protein holds a special place. Naturally found in certain viruses, this enzyme recognises and modifies specific genetic sequences, creating changes that switch on or off whatever gene is close by. Genetically engineering cells or organisms so that they carry Cre and its target sequences allows scientists to control the activation of a given gene, often in a single tissue or organ. However, this relies on the ability to activate the Cre protein ‘on demand’ once it is in the cells of interest. One way to do so is to split the enzyme into two pieces, which can then reassemble when exposed to blue light. Yet, this involves the challenging step of introducing both parts separately into a tissue. Instead, Duplus-Bottin et al. engineered LiCre, a new system where a large section of the Cre protein is fused to a light sensor used by oats to detect their environment. LiCre is off in the dark, but it starts to recognize and modify Cre target sequences when exposed to blue light. Duplus-Bottin et al. then assessed how LiCre compares to the two-part Cre system in baker's yeast and human kidney cells. This showed that the new protein is less ‘incorrectly’ active in the dark, and can switch on faster under blue light. The improved approach could give scientists a better tool to study the role of certain genes at precise locations and time points, but also help them to harness genetic sequences for industry or during gene therapy.
Identifiants
pubmed: 33620312
doi: 10.7554/eLife.61268
pii: 61268
pmc: PMC7997657
doi:
pii:
Substances chimiques
Saccharomyces cerevisiae Proteins
0
Cre recombinase
EC 2.7.7.-
Integrases
EC 2.7.7.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Informations de copyright
© 2021, Duplus-Bottin et al.
Déclaration de conflit d'intérêts
HD, MS, GY A patent application covering LiCre and its potential applications has been filed. Ref: FR3079832 A1 and WO2019193205. Patent applicant: CNRS; inventors: Hélène Duplus-Bottin, Martin Spichty and Gaël Yvert. GT, CP, PM, TO, FV No competing interests declared
Références
Nucleic Acids Res. 2014 Apr;42(6):3894-907
pubmed: 24413561
Yeast. 2015 Dec;32(12):711-20
pubmed: 26305040
Nat Commun. 2020 May 1;11(1):2141
pubmed: 32358538
Chembiochem. 2018 Jun 18;19(12):1244-1249
pubmed: 29701891
Elife. 2017 Jul 06;6:
pubmed: 28682236
Proc Natl Acad Sci U S A. 2015 Jan 6;112(1):112-7
pubmed: 25535392
Science. 2003 Sep 12;301(5639):1541-4
pubmed: 12970567
ACS Chem Biol. 2014 Mar 21;9(3):617-21
pubmed: 24428544
Trends Biotechnol. 2016 Aug;34(8):652-664
pubmed: 26996613
Nat Chem Biol. 2016 Jun;12(6):425-30
pubmed: 27065233
Sci Signal. 2011 Aug 2;4(184):ra50
pubmed: 21868352
Nat Chem Biol. 2009 Nov;5(11):827-34
pubmed: 19718042
Nature. 1997 Sep 4;389(6646):40-6
pubmed: 9288963
BMC Bioinformatics. 2009 Apr 09;10:106
pubmed: 19358741
Curr Opin Cell Biol. 2014 Oct;30:112-20
pubmed: 25216352
Biochem Biophys Res Commun. 2020 May 21;526(1):213-217
pubmed: 32204914
Chem Biol. 2013 Apr 18;20(4):619-26
pubmed: 23601651
Curr Genet. 1991 Nov;20(5):365-72
pubmed: 1807826
Yeast. 2007 May;24(5):447-55
pubmed: 17315265
Biochemistry. 2002 Jun 11;41(23):7183-9
pubmed: 12044148
Biotechnol Bioeng. 2008 Feb 15;99(3):666-77
pubmed: 17705244
Nucleic Acids Res. 2004 Jul 12;32(12):e102
pubmed: 15249598
J Comput Chem. 2008 Apr 15;29(5):701-15
pubmed: 17918282
ACS Synth Biol. 2020 Feb 21;9(2):227-235
pubmed: 31961670
Small. 2018 Jul;14(30):e1800543
pubmed: 29968382
J Mol Graph. 1996 Feb;14(1):33-8, 27-8
pubmed: 8744570
J Chem Theory Comput. 2016 Jan 12;12(1):405-13
pubmed: 26631602
Nucleic Acids Res. 2003 Sep 15;31(18):5449-60
pubmed: 12954782
Microb Cell Fact. 2014 Jan 21;13:12
pubmed: 24443802
Nucleic Acids Res. 2019 Sep 26;47(17):e97
pubmed: 31287871
Metab Eng. 2015 Mar;28:8-18
pubmed: 25475893
Appl Environ Microbiol. 2007 Jul;73(13):4342-50
pubmed: 17496128
Chem Commun (Camb). 2016 Jun 30;52(55):8529-32
pubmed: 27277957
Proc Natl Acad Sci U S A. 2001 Mar 13;98(6):2995-3000
pubmed: 11248020
Microbiol Spectr. 2015 Feb;3(1):MDNA3-0014-2014
pubmed: 26104563
J Comput Chem. 2009 Jul 30;30(10):1545-614
pubmed: 19444816
Chembiochem. 2010 Mar 22;11(5):653-63
pubmed: 20187057
Mol Pharmacol. 2007 Oct;72(4):812-25
pubmed: 17596375
Bioconjug Chem. 2012 Sep 19;23(9):1945-51
pubmed: 22917215
Nat Methods. 2012 Mar 04;9(4):379-84
pubmed: 22388287
ACS Synth Biol. 2019 Oct 18;8(10):2359-2371
pubmed: 31592660
J Biol Chem. 2001 Sep 28;276(39):36493-500
pubmed: 11443119
Trends Neurosci. 2001 May;24(5):251-4
pubmed: 11311363
Nat Commun. 2014 Jul 14;5:4404
pubmed: 25019686
Front Mol Biosci. 2015 May 12;2:18
pubmed: 25988185
J Clin Invest. 1996 Aug 1;98(3):600-3
pubmed: 8698848
Nat Commun. 2018 May 22;9(1):1931
pubmed: 29789561
Nat Methods. 2010 Dec;7(12):973-5
pubmed: 21037589
Nat Chem Biol. 2016 Dec;12(12):1059-1064
pubmed: 27723747
Acta Biochim Pol. 2005;52(2):541-4
pubmed: 15912212
Yeast. 2010 Dec;27(12):983-98
pubmed: 20632327
Dis Model Mech. 2009 Sep-Oct;2(9-10):508-15
pubmed: 19692579
Biotechnol Bioeng. 2016 Jan;113(1):72-81
pubmed: 26108688
J Am Chem Soc. 2005 Sep 28;127(38):13088-9
pubmed: 16173704
Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10887-90
pubmed: 8855277
Nucleic Acids Res. 2003 Nov 1;31(21):e131
pubmed: 14576331
Nat Methods. 2017 Apr;14(4):391-394
pubmed: 28288123
Nat Commun. 2019 Oct 24;10(1):4845
pubmed: 31649244
Front Genet. 2018 Nov 02;9:518
pubmed: 30450113