CRISPR-Cas9 homology-independent targeted integration of exons 1–19 restores full-length dystrophin in mice

Stephenson, Anthony A.; Nicolau, Stefan; Vetter, Tatyana A.; Dufresne, Gabrielle P.; Frair, Emma C.; Sarff, Jessica E.; Wheeler, Gregory L.; Kelly, Benjamin J.; White, Peter; Flanigan, Kevin M.

doi:10.1016/j.omtm.2023.08.009

Cited by 2 publications

(1 citation statement)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Indeed, in our hands biGBac is the cloning system allowing the easiest assembly of a large number of genetic modules. Moreover, genomic integration though HITI after a Cas9 double strand break is the most efficient reported approach for knocking in foreign DNA into the genome of any cell types 4,36 .…”

Section: Introductionmentioning

confidence: 99%

biGMamAct: efficient CRISPR/Cas9-mediated docking of large functional DNA cargoes at theACTBlocus

Pelosse,

Marcia

2024

Preprint

View full text Add to dashboard Cite

Recent advances in molecular and cell biology and imaging have unprecedentedly enabled multi-scale structure-functional studies of entire metabolic pathways from atomic to micrometer resolution, and the visualization of macromolecular complexes in situ, especially if these molecules are expressed with appropriately-engineered and easily-detectable tags. However, genome editing in eukaryotic cells is challenging when generating stable cell lines loaded with large DNA cargoes. To address this limitation, here, we have conceived biGMamAct, a system that allows the straightforward assembly of a multitude of genetic modules and their subsequent integration in the genome at the ACTB locus with high efficacy, through standardized cloning steps. Our technology encompasses a set of modular plasmids for mammalian expression, which can be efficiently docked into the genome in tandem with a validated Cas9/sgRNA pair through homologous-independent targeted insertion (HITI). As a proof of concept, we have generated a stable cell line loaded with an 18.3-kilobase-long DNA cargo to express 6 fluorescently-tagged proteins and simultaneously visualize 5 different subcellular compartments. Our protocol leads from the in-silico design to the genetic and functional characterization of single clones within 6 weeks and can be implemented by any researcher with familiarity with molecular biology and access to mammalian cell culturing infrastructure.

show abstract

Section: Introductionmentioning

confidence: 99%