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

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.