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Sofie Andersen

CRISPR-Cas9-directed gene tagging using a single integrase-defective lentiviral vector carrying a transposase-based Cas9 off switch

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avis › Tidsskriftartikel › Forskning › peer review

DOI

Locus-directed DNA cleavage induced by the CRISPR-Cas9 system triggers DNA repair mechanisms allowing gene repair or targeted insertion of foreign DNA. For gene insertion to be successful, availability of a homologous donor template needs to be timed with cleavage of the DNA by the Cas9 endonuclease guided by a target-specific single guide RNA (sgRNA). We present a novel approach for targeted gene insertion based on a single integrase-defective lentiviral vector (IDLV) carrying a Cas9 off switch. Gene insertion using this approach benefits from transposon-based stable Cas9 expression, which is switched off by excision-only transposase protein co-delivered in IDLV particles carrying a combined sgRNA/donor vector. This one-vector approach supports potent (up to >80%) knockin of a full-length EGFP gene sequence. This traceless cell engineering method benefits from high stable levels of Cas9, timed intracellular availability of the molecular tools, and a built-in feature to turn off Cas9 expression after DNA cleavage. The simple technique is based on transduction with a single IDLV, which holds the capacity to transfer larger donor templates, allowing robust gene knockin or tagging of genes in a single step.

OriginalsprogEngelsk
TidsskriftMolecular Therapy - Nucleic Acids
Vol/bind29
Sider (fra-til)563-576
ISSN2162-2531
DOI
StatusUdgivet - sep. 2022

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