CRISPR-Trap: a clean approach for the generation of gene knockouts and gene replacements in human cells

Conventional nonhomologous end joining–based gene knockouts can lead to the production of C-terminally truncated proteins with potentially residual or dominant negative functions. Combining CRISPR/Cas9 with gene traps targeting the first intron (CRISPR-Trap), however, completely prevents the expression of the open reading frame, resulting in clean gene knockouts.

Inducible Gene Trapping with Drug-Selectable Markers and Cre/loxP To Identify Developmentally Regulated Genes

ABSTRACT Gene trapping in mouse embryonic stem cells is an important genetic approach that allows simultaneous mutation of genes and generation of corresponding mutant mice. We designed a selection scheme with drug selection markers and Cre/loxP technology which allows screening of gene trap events that responded to a signaling molecule in a 96-well format. Nine hundred twenty gene trap clones were assayed, and 258 were classified as gene traps induced by in vitro differentiation. Sixty-five of the in vitro differentiation-inducible gene traps were also responsive to retinoic acid treatment. In vivo analysis revealed that 85% of the retinoic acid-inducible gene traps trapped developmentally regulated genes, consistent with the observation that genes induced by retinoic acid treatment are likely to be developmentally regulated. Our results demonstrate that the inducible gene trapping system described here can be used to enrich in vitro for traps in genes of interest. Furthermore, we demonstrate that the cre reporter is extremely sensitive and can be used to explore chromosomal regions that are not detectable with neo as a selection cassette.