TY - JOUR
T1 - Nanoblades allow high-level genome editing in murine and human organoids
AU - Tiroille, Victor
AU - Krug, Adrien
AU - Bokobza, Emma
AU - Kahi, Michel
AU - Bulcaen, Mattijs
AU - Ensinck, Marjolein M
AU - Geurts, Maarten H
AU - Hendriks, Delilah
AU - Vermeulen, François
AU - Larbret, Frédéric
AU - Gutierrez-Guerrero, Alejandra
AU - Chen, Yu
AU - Van Zundert, Indra
AU - Rocha, Susana
AU - Rios, Anne C
AU - Medaer, Louise
AU - Gijsbers, Rik
AU - Mangeot, Philippe E
AU - Clevers, Hans
AU - Carlon, Marianne S
AU - Bost, Frédéric
AU - Verhoeyen, Els
N1 - © 2023 Institut national de la santé et de la recherche médicale.
PY - 2023/9/12
Y1 - 2023/9/12
N2 - Genome engineering has become more accessible thanks to the CRISPR-Cas9 gene-editing system. However, using this technology in synthetic organs called "organoids" is still very inefficient. This is due to the delivery methods for the CRISPR-Cas9 machinery, which include electroporation of CRISPR-Cas9 DNA, mRNA, or ribonucleoproteins containing the Cas9-gRNA complex. However, these procedures are quite toxic for the organoids. Here, we describe the use of the "nanoblade (NB)" technology, which outperformed by far gene-editing levels achieved to date for murine- and human tissue-derived organoids. We reached up to 75% of reporter gene knockout in organoids after treatment with NBs. Indeed, high-level NB-mediated knockout for the androgen receptor encoding gene and the cystic fibrosis transmembrane conductance regulator gene was achieved with single gRNA or dual gRNA containing NBs in murine prostate and colon organoids. Likewise, NBs achieved 20%-50% gene editing in human organoids. Most importantly, in contrast to other gene-editing methods, this was obtained without toxicity for the organoids. Only 4 weeks are required to obtain stable gene knockout in organoids and NBs simplify and allow rapid genome editing in organoids with little to no side effects including unwanted insertion/deletions in off-target sites thanks to transient Cas9/RNP expression.
AB - Genome engineering has become more accessible thanks to the CRISPR-Cas9 gene-editing system. However, using this technology in synthetic organs called "organoids" is still very inefficient. This is due to the delivery methods for the CRISPR-Cas9 machinery, which include electroporation of CRISPR-Cas9 DNA, mRNA, or ribonucleoproteins containing the Cas9-gRNA complex. However, these procedures are quite toxic for the organoids. Here, we describe the use of the "nanoblade (NB)" technology, which outperformed by far gene-editing levels achieved to date for murine- and human tissue-derived organoids. We reached up to 75% of reporter gene knockout in organoids after treatment with NBs. Indeed, high-level NB-mediated knockout for the androgen receptor encoding gene and the cystic fibrosis transmembrane conductance regulator gene was achieved with single gRNA or dual gRNA containing NBs in murine prostate and colon organoids. Likewise, NBs achieved 20%-50% gene editing in human organoids. Most importantly, in contrast to other gene-editing methods, this was obtained without toxicity for the organoids. Only 4 weeks are required to obtain stable gene knockout in organoids and NBs simplify and allow rapid genome editing in organoids with little to no side effects including unwanted insertion/deletions in off-target sites thanks to transient Cas9/RNP expression.
U2 - 10.1016/j.omtn.2023.06.004
DO - 10.1016/j.omtn.2023.06.004
M3 - Article
C2 - 37435135
SN - 2162-2531
VL - 33
SP - 57
EP - 74
JO - Molecular therapy. Nucleic acids
JF - Molecular therapy. Nucleic acids
ER -