Nanoblades allow high-level genome editing in murine and human organoids

被引:9
|
作者
Tiroille, Victor [1 ]
Krug, Adrien [1 ]
Bokobza, Emma [1 ]
Kahi, Michel [1 ]
Bulcaen, Mattijs [3 ,4 ]
Ensinck, Marjolein M. [3 ,4 ]
Geurts, Maarten H. [5 ,6 ,7 ]
Hendriks, Delilah [5 ,6 ,7 ]
Vermeulen, Francois [8 ]
Larbret, Frederic [1 ]
Gutierrez-Guerrero, Alejandra [2 ]
Chen, Yu [9 ,10 ]
Van Zundert, Indra [11 ]
Rocha, Susana [12 ]
Rios, Anne C. [13 ]
Medaer, Louise [3 ]
Gijsbers, Rik [3 ]
Mangeot, Philippe E. [2 ]
Clevers, Hans [5 ,6 ,7 ,15 ]
Carlon, Marianne S.
Bost, Frederic [1 ,14 ]
Verhoeyan, Els [1 ,2 ]
机构
[1] Univ Cote Azur, INSERM, C3M, F-06204 Nice, France
[2] Univ Lyon, Univ Claude Bernard Lyon 1, Ecole Normale Super Lyon,UMR5308, CIRI Int Ctr Infectiol Res,U1111,Inserm,CNRS, F-69007 Lyon, France
[3] Katholieke Univ Leuven, Fac Med, Dept Pharmaceut & Pharmacol Sci, Lab Mol Virol & Gene Therapy, Leuven, Belgium
[4] Katholieke Univ Leuven, Dept Chron Dis & Metab, Lab Resp Dis & Thorac Surg BREATHE, Leuven, Belgium
[5] Royal Netherlands Acad Arts & Sci KNAW, Hubrecht Inst, Utrecht, Netherlands
[6] Univ Med Ctr Utrecht, Utrecht, Netherlands
[7] Hubrecht Inst, Oncode Inst, Utrecht, Netherlands
[8] UZ Leuven, Dept Pediat, Leuven, Belgium
[9] Mem Sloan, Dept Med, Human Oncol & Pathogenesis Program, New York, NY 10065 USA
[10] Weill Cornell Med Coll, Dept Med, New York, NY 10065 USA
[11] Eindhoven Univ Technol, Dept Biomed Engn, Synthet Biol Grp, POB 513, NL-5600 MB Eindhoven, Netherlands
[12] Katholieke Univ Leuven, Dept Chem, Mol Imaging & Photon, Celestijnenlaan 200F, B-3001 Heverlee, Belgium
[13] Princess Maxima Ctr Pediat Oncol, Utrecht, Netherlands
[14] Equipe Labelisee Ligue Natl Canc, Toulouse, France
[15] Roche, Pharma Res Early Dev, Basel, Switzerland
来源
关键词
Gene editing; CRISPR; Cas9; nanoblade; organoid; knock-out; CFTR; colon organoid; prostate organoid; androgen receptor; virus like particle; HUMAN INTESTINAL ORGANOIDS; STEM-CELL ORGANOIDS; GENE-TRANSFER; T-CELLS; CRISPR; CFTR; IDENTIFICATION; DISEASE; REPAIR; ADULT;
D O I
10.1016/j.omtn.2023.06.004
中图分类号
R-3 [医学研究方法]; R3 [基础医学];
学科分类号
1001 ;
摘要
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' 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 knock-out in organoids after treatment with nanoblades. Indeed, high-level nanoblade-mediated knock-out for the androgen receptor encoding gene and the cystic fibrosis transmembrane conductance regulator gene was achieved with single gRNA or dual gRNA containing nanoblades in murine prostate and colon organoids. Likewise, nanoblades achieved 20% to 50% gene editing in human organoids. Most importantly, in contrast to other gene editing methods, this was obtained without toxicity for the organoids. Only four weeks are required to obtain stable gene knock-out in organoids and nanoblades 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.
引用
收藏
页码:57 / 74
页数:51
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