PT  - JOURNAL ARTICLE
AU  - Megan D. Schertzer
AU  - Eliza Thulson
AU  - Keean C.A. Braceros
AU  - David M. Lee
AU  - Emma R. Hinkle
AU  - Ryan M. Murphy
AU  - Susan O. Kim
AU  - Eva C.M. Vitucci
AU  - J. Mauro Calabrese
TI  - A piggyBac-based toolkit for inducible genome editing in mammalian cells
AID  - 10.1101/448803
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 448803
4099  - http://biorxiv.org/content/early/2018/10/20/448803.short
4100  - http://biorxiv.org/content/early/2018/10/20/448803.full
AB  - We describe the development and application of a novel series of vectors that facilitate CRISPR-Cas9-mediated genome editing in mammalian cells, which we call CRISPR-Bac. CRISPR-Bac leverages the piggyBac transposon to randomly insert CRISPR-Cas9 components into mammalian genomes. In CRISPR-Bac, a single piggyBac cargo vector containing a doxycycline-inducible Cas9 or catalytically-dead Cas9 (dCas9) variant and a gene conferring resistance to Hygromycin B is co-transfected with a plasmid expressing the piggyBac transposase. A second cargo vector, expressing a single-guide RNA (sgRNA) of interest, the reverse-tetracycline TransActivator (rtTA), and a gene conferring resistance to G418, is also cotransfected. Subsequent selection on Hygromycin B and G418 generates polyclonal cell populations that stably express Cas9, rtTA, and the sgRNA(s) of interest. Using Mus musculus-derived embryonic and trophoblast stem cells, we show that CRISPR-Bac can be used to knockdown proteins of interest, to create targeted genetic deletions with high efficiency, and to activate or repress transcription of protein-coding genes and an imprinted long noncoding RNA. The ratio of sgRNA-to-Cas9-to-transposase can be adjusted in transfections to alter the average number of cargo insertions into the genome. sgRNAs targeting multiple genes can be inserted in a single transfection. CRISPR-Bac is a versatile platform for genome editing that simplifies the generation of mammalian cells that stably express the CRISPR-Cas9 machinery.