ATAD3A Gene Knockout via CRISPR-Cas9

Single guide RNAs targeting ATAD3A (oligonucleotides used given in Table 1) were designed using the CRISPOR tool (crispor.tefor.net) and cloned into lentiviral construct lentiCRISPRv2-hygro, a gift from Brett Stringer (Flinders Health and Medical Research Institute, Adelaide, Australia; Stringer et al., 2019 (link); plasmid #98291; Addgene), following the protocol provided on the plasmid website. EVs 3 and 12 are two clones obtained after BSMBI digestion to remove buffer sequence, blunting and ligation using the Quick blunting kit (New England Biolabs), following the manufacturer’s instructions. Lentiviral particles were produced, and THP-1 cells transduced as described for shRNA. 2 d after transduction, transduced cells were selected with 500 µg/ml hygromycin B (Invivogen), and selected cell pools were analyzed 7 d after transduction.

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Lepelley A., Della Mina E., Van Nieuwenhove E., Waumans L., Fraitag S., Rice G.I., Dhir A., Frémond M.L., Rodero M.P., Seabra L., Carter E., Bodemer C., Buhas D., Callewaert B., de Lonlay P., De Somer L., Dyment D.A., Faes F., Grove L., Holden S., Hully M., Kurian M.A., McMillan H.J., Suetens K., Tyynismaa H., Chhun S., Wai T., Wouters C., Bader-Meunier B, & Crow Y.J. (2021). Enhanced cGAS-STING–dependent interferon signaling associated with mutations in ATAD3A. The Journal of Experimental Medicine, 218(10), e20201560.

Publication 2021

Quick blunting kit hygromycin B

Buffer Cell Clones Digestion Hygromycin b Ligation Oligonucleotides Plasmid Shrna Single guide rnas Thp 1 cells

Corresponding Organization : MRC Institute of Genetics and Molecular Medicine

Other organizations : VIB-KU Leuven Center for Brain & Disease Research, Universitair Ziekenhuis Leuven, KU Leuven, Assistance Publique – Hôpitaux de Paris, Hôpital Necker-Enfants Malades, University of Manchester, Manchester Academic Health Science Centre, McGill University Health Centre, McGill University, Ghent University Hospital, Ghent University, Institut Necker Enfants Malades, Rega Institute for Medical Research, Children's Hospital of Eastern Ontario, West Suffolk NHS Foundation Trust, West Suffolk Hospital, Addenbrooke's Hospital, University College London, University of Ottawa, University of Helsinki, Sorbonne Paris Cité, Délégation Paris 5, Institut Pasteur

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Variable analysis

independent variables

Guide RNAs targeting ATAD3A

dependent variables

Transduction efficiency of THP-1 cells

control variables

Hygromycin B concentration used for selection of transduced cells

controls

Positive control: None mentioned
Negative control: None mentioned

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