TY - JOUR
T1 - Gene editing in hematopoietic stem cells by co-delivery of Cas9/sgRNA ribonucleoprotein and templates for homology-directed repair in 'all-in-one' lentivirus-derived nanoparticles
AU - Andersen, Sofie
AU - Wolff, Jonas Holst
AU - Skov, Thomas Wisbech
AU - Janns, Jacob Hørlück
AU - Davis, Luther John
AU - Haldrup, Jakob H
AU - Haslund, Didde
AU - Revenfeld, Anne Louise
AU - Relkovic, Dinko
AU - Møller, Bjarne K
AU - Lund, Jacob
AU - Bak, Rasmus O
AU - Thomsen, Emil Aagaard
AU - Mikkelsen, Jacob Giehm
N1 - © The Author(s) 2025. Published by Oxford University Press.
PY - 2025/8/28
Y1 - 2025/8/28
N2 - Repair of double-strand DNA breaks generated by site-directed endonucleases, like Cas9, is the hallmark of gene editing based on homology-directed repair (HDR). HDR uses an exogenous DNA template to restore the cleaved DNA sequence and can facilitate specific gene corrections as well as insertion of genes or partial complementary DNA (cDNA) sequences. For CRISPR/Cas-directed gene editing, co-administration of the Cas9/single guide RNA (sgRNA) ribonucleoprotein (RNP) complex and a DNA template typically involves two different delivery strategies or different types of vehicles. This requires exquisite timing of delivery and may potentially challenge safety and therapeutic applicability. There is a need therefore for technologies that can ferry complete editing tool kits into cells. Here, we demonstrate the use of lentivirus-derived nanoparticles (LVNPs) to transport both RNP complexes and vector RNA, which upon reverse transcription serves as a repair template for HDR-directed gene editing. Such 'all-in-one' LVNPs support targeted gene insertion with reduced off-target effects relative to nucleofection procedures. We show potent editing in the HBB gene in human erythroid progenitor cells as well as HDR-directed editing in hematopoietic stem and progenitor cells. Our findings mark a first step toward using a single virus-derived vehicle for delivering a full HDR gene editing kit.
AB - Repair of double-strand DNA breaks generated by site-directed endonucleases, like Cas9, is the hallmark of gene editing based on homology-directed repair (HDR). HDR uses an exogenous DNA template to restore the cleaved DNA sequence and can facilitate specific gene corrections as well as insertion of genes or partial complementary DNA (cDNA) sequences. For CRISPR/Cas-directed gene editing, co-administration of the Cas9/single guide RNA (sgRNA) ribonucleoprotein (RNP) complex and a DNA template typically involves two different delivery strategies or different types of vehicles. This requires exquisite timing of delivery and may potentially challenge safety and therapeutic applicability. There is a need therefore for technologies that can ferry complete editing tool kits into cells. Here, we demonstrate the use of lentivirus-derived nanoparticles (LVNPs) to transport both RNP complexes and vector RNA, which upon reverse transcription serves as a repair template for HDR-directed gene editing. Such 'all-in-one' LVNPs support targeted gene insertion with reduced off-target effects relative to nucleofection procedures. We show potent editing in the HBB gene in human erythroid progenitor cells as well as HDR-directed editing in hematopoietic stem and progenitor cells. Our findings mark a first step toward using a single virus-derived vehicle for delivering a full HDR gene editing kit.
UR - https://www.scopus.com/pages/publications/105013344167
U2 - 10.1093/nar/gkaf767
DO - 10.1093/nar/gkaf767
M3 - Journal article
C2 - 40808298
SN - 0305-1048
VL - 53
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 15
M1 - gkaf767
ER -