Lauritsen, Ida

Martinez, Virginia

Nørholm, Morten H.H.

Wolff, Tonja

Online Research Database In Technology

                       General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.  • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal   If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.     Downloaded from orbit.dtu.dk on: Nov 08, 2017Conditional protein depletion using small degradation tags and CRISPR/Cas9 systemsMartinez, Virginia; Lauritsen, Ida; Hobel, Tonja; Nørholm, MortenPublished in:Book of Abstracts. DTU's Sustain Conference 2015Publication date:2015Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Martinez, V., Lauritsen, I., Wolff, T., & Nørholm, M. H. H. (2015). Conditional protein depletion using smalldegradation tags and CRISPR/Cas9 systems. In Book of Abstracts. DTU's Sustain Conference 2015 [B-4]Lyngby: Technical University of Denmark (DTU).			Conditional	protein	depletion	using	small	degradation	tags	and	CRISPR/Cas9	systems			Virginia	Martínez1,	Ida	Lauritsen1,	Tonja	Wolff1	and	Morten	H.	H.	Nørholm*1	1:	DTU	Biosustain	*Corresponding	author	email:	morno@biosustain.dtu.dk		In	 the	 last	 decade,	 metabolically	 engineered	 microorganisms	 have	 been	 developed	 for	 the	 bio-based	production	of	chemicals,	fuels,	bioactive	compounds,	proteins	or	materials.	For	these	purposes,	metabolic	engineers	 need	 a	 toolset	 to	 accurately	 control	 the	 native	 and	 heterologous	 reactions	 inside	 the	 cell,	 by	manipulating	the	 involved	enzymes1.	However,	selective	post-translational	 inhibition	of	enzymatic	activity	is	still	challenging	since	proteins	prevail	in	cells	for	much	longer	than	their	transcription	or	translation	has	been	inhibited.		Here,	we	provide	a	conditional	protein	knockdown	technology	that	allows	the	in	vivo	regulation	of	protein	abundance	 inside	 the	 cells.	 This	 strategy,	 termed	 “Protein	 interference”	 (PROTi),	 is	 based	 on	 the	 use	 of	small	 degradation	 tags	 (N-degrons)2	 that	 can	 be	 integrated	 by	 CRISPR/Cas9-based	 genome	 editing,	targeting	proteins	for	degradation.	Further,	combination	of	the	PROTi	technology	with	gene	repression	by	CRISPRi	 system3,	 accelerated	 cellular	 depletion	 of	 the	 targeted	 proteins.	 This	 technology	 provides	 a	valuable	 tool	 for	 balancing	 cellular	 pathways	 at	 the	 post-translational	 level	 for	 metabolic	 engineering	purposes,	for	studying	essential	proteins	or	for	discovering	novel	antibiotic	targets.			1.	Holtz,	W.J.	 and	Keasling,	 J.D.	 2010.	 Engineering	 static	 and	dynamic	 control	of	 synthetic	pathways.	Cell	140:	19–23.	2.	 Erbse,	 A.	 et	 al.,	 2006.	 ClpS	 is	 an	 essential	 component	 of	 the	 N-end	 rule	 pathway	 in	 Escherichia	 coli.	Nature	439:	753–756.	3.	Qi,	L.S.	et	al.,	2013.	Repurposing	CRISPR	as	an	RNA-guided	platform	for	sequence-specific	control	of	gene	expression.	Cell	152:	1173–1183.		Sustain DTU Abstract: B-4

Conditional protein depletion using small degradation tags and CRISPR/Cas9 systems

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Conditional protein depletion using small degradation tags and CRISPR/Cas9 systems

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