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

TY - JOUR

T1 - CRISPR-Cas9-directed gene tagging using a single integrase-defective lentiviral vector carrying a transposase-based Cas9 off switch

AU - Thomsen, Emil Aagaard

AU - Skipper, Kristian Alsbjerg

AU - Andersen, Sofie

AU - Haslund, Didde

AU - Skov, Thomas Wisbech

AU - Mikkelsen, Jacob Giehm

N1 - Publisher Copyright: © 2022 The Author(s)

PY - 2022/9

Y1 - 2022/9

N2 - 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.

AB - 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.

KW - AAV

KW - CRISPR-Cas9

KW - DNA transposon

KW - Donor template

KW - Gene tagging

KW - HDR

KW - IDLV

KW - lentivirus

KW - MT: Delivery Strategies

KW - piggyBac

KW - protein delivery

U2 - 10.1016/j.omtn.2022.08.005

DO - 10.1016/j.omtn.2022.08.005

M3 - Journal article

C2 - 36090759

AN - SCOPUS:85136182555

SN - 2162-2531

VL - 29

SP - 563

EP - 576

JO - Molecular Therapy - Nucleic Acids

JF - Molecular Therapy - Nucleic Acids

ER -