title: Application of CRISPR/Cas9 to Autophagy Research.
abstract: The ability to efficiently modulate autophagy activity is paramount in the study of the field. Conventional broad-range autophagy inhibitors and genetic manipulation using RNA interference (RNAi), although widely used in autophagy research, are often limited in specificity or efficacy. In this chapter, we address the problems of conventional autophagy-modulating tools by exploring the use of three different CRISPR/Cas9 systems to abrogate autophagy in numerous human and mouse cell lines. The first system generates cell lines constitutively deleted of ATG5 or ATG7 whereas the second and third systems express a Tet-On inducible-Cas9 that enables regulated deletion of ATG5 or ATG7. We observed the efficiency of autophagy inhibition using the CRISPR/Cas9 strategy to surpass that of RNAi, and successfully generated cells with complete and sustained autophagy disruption through the CRISPR/Cas9 technology.
authors.affiliation: Cancer Research UK Beatson Institute, Glasgow, United Kingdom.
authors.forename: J
authors.initials: J
authors.lastname: O'Prey
authors.affiliation: Cancer Research UK Beatson Institute, Glasgow, United Kingdom.
authors.forename: J
authors.initials: J
authors.lastname: Sakamaki
authors.affiliation: Cancer Research UK Beatson Institute, Glasgow, United Kingdom.
authors.forename: A D
authors.initials: AD
authors.lastname: Baudot
authors.affiliation: The Francis Crick Institute, London, United Kingdom.
authors.forename: M
authors.initials: M
authors.lastname: New
authors.affiliation: The Francis Crick Institute, London, United Kingdom.
authors.forename: T
authors.initials: T
authors.lastname: Van Acker
authors.affiliation: The Francis Crick Institute, London, United Kingdom.
authors.forename: S A
authors.initials: SA
authors.lastname: Tooze
authors.affiliation: Cancer Research UK Beatson Institute, Glasgow, United Kingdom. Electronic address: j.long@beatson.gla.ac.uk.
authors.forename: J S
authors.initials: JS
authors.lastname: Long
authors.affiliation: Cancer Research UK Beatson Institute, Glasgow, United Kingdom. Electronic address: k.ryan@beatson.gla.ac.uk.
authors.forename: K M
authors.initials: KM
authors.lastname: Ryan
mesh_terms: D000818:Animals; D001343:Autophagy; D000071187:Autophagy-Related Protein 5 / Q000235:genetics; D000071193:Autophagy-Related Protein 7 / Q000235:genetics; D064113:CRISPR-Cas Systems; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D000072669:Gene Editing / Q000379:methods*; D006801:Humans; D051379:Mice; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics
chemical_list: D000071187:Autophagy-Related Protein 5; D000094704:RNA, Guide, CRISPR-Cas Systems; D000071193:Autophagy-Related Protein 7
keywords: ATG; Autophagy; CRISPR; Cas9; RNAi