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.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
O'Prey J,Sakamaki J,Baudot AD,New M,Van Acker T,Tooze SA,Long JS,Ryan KMdoi
10.1016/bs.mie.2016.09.076subject
Has Abstractpub_date
2017-01-01 00:00:00pages
79-108eissn
0076-6879issn
1557-7988pii
S0076-6879(16)30349-4journal_volume
588pub_type
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