A protocol for efficient CRISPR-Cas9-mediated knock-in in colorectal cancer patient-derived organoids

Okamoto, Takuya; Natsume, Yasuko; Yamanaka, Hitomi; Fukuda, Mayuko; Yao, Ryoji

doi:10.1016/j.xpro.2021.100780

Cited by 12 publications

(6 citation statements)

References 6 publications

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“…Despite this revolutionary success, there remains an urgent need for increased simplicity and efficiency of genome editing in research organisms. Protocols for efficient knock-in in cultured organoids [ 179 ] and Japanese rice fish [ 180 ] have succeeded, yet nothing has been reported in mammals to this point. Moreover, previous studies were undertaken that demonstrated a synergistic interaction between the transcription factors NFAT and MEF-2 in regulating MB gene expression patterns in the muscle [ 181 ].…”

Section: Potential Therapeutic Implications Of Mb In Bat-mediated Ene...mentioning

confidence: 99%

Myoglobin in Brown Adipose Tissue: A Multifaceted Player in Thermogenesis

Aboouf,

Gorr,

Hamdy

et al. 2023

Cells

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Brown adipose tissue (BAT) plays an important role in energy homeostasis by generating heat from chemical energy via uncoupled oxidative phosphorylation. Besides its high mitochondrial content and its exclusive expression of the uncoupling protein 1, another key feature of BAT is the high expression of myoglobin (MB), a heme-containing protein that typically binds oxygen, thereby facilitating the diffusion of the gas from cell membranes to mitochondria of muscle cells. In addition, MB also modulates nitric oxide (NO•) pools and can bind C16 and C18 fatty acids, which indicates a role in lipid metabolism. Recent studies in humans and mice implicated MB present in BAT in the regulation of lipid droplet morphology and fatty acid shuttling and composition, as well as mitochondrial oxidative metabolism. These functions suggest that MB plays an essential role in BAT energy metabolism and thermogenesis. In this review, we will discuss in detail the possible physiological roles played by MB in BAT thermogenesis along with the potential underlying molecular mechanisms and focus on the question of how BAT–MB expression is regulated and, in turn, how this globin regulates mitochondrial, lipid, and NO• metabolism. Finally, we present potential MB-mediated approaches to augment energy metabolism, which ultimately could help tackle different metabolic disorders.

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Section: Potential Therapeutic Implications Of Mb In Bat-mediated Ene...mentioning

confidence: 99%

Myoglobin in Brown Adipose Tissue: A Multifaceted Player in Thermogenesis

Aboouf,

Gorr,

Hamdy

et al. 2023

Cells

View full text Add to dashboard Cite

show abstract

“…Here, we have shown an effective delivery methodology for the CRISPR-Cas9 system called “nanoblades,” which permitted to obtain high levels of gene editing (20%–80%) in human and mouse organoids without the requirement for enrichment via drug selection or fluorescent reporter isolation. 33 These are levels of gene editing that are otherwise exclusively achieved in iPSC-derived organoids. 34 Brain organoids, for example, are solely derived from iPSCs and since gene editing is performed at the level of the iPSCs, which is highly efficient, deleting genes or introducing genes (e.g., oncogenes) can be performed with ease.…”

Section: Discussionmentioning

confidence: 99%

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

Tiroille¹,

Krug²,

Bokobza³

et al. 2023

Molecular Therapy - Nucleic Acids

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“…Here we have shown a novel delivery methodology for the CRISPR/Cas9 system called 'nanoblades', which permitted to obtain high levels of gene editing (20%-80%) in human and mouse organoids without the requirement for enrichment via drug selection or fluorescent reporter isolation (Okamoto et al, 2021). These are levels of gene editing that are otherwise exclusively achieved in induced pluripotent stem cells (IPSC ) derived organoids (Son et al, 2022).…”

Section: Discussionmentioning

confidence: 99%

“…The method uses a donor template coding for an expression cassette driving a fluorescent protein such as GFP flanked by homologous genome sequences (homology arms). Through recombination at the DSB induced by Cas9, this template permitted easy identification of KO/knock-in organoids through GFP expression (Okamoto et al, 2021). Alternatively, a NHEJ strategy relying on the piggybac-transposase system to integrate hygromycin/GFP was used to screen for fluorescence or drug resistance (Artegiani et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

Nanoblades allow high-level genome editing in organoids

Tirolle

Krug

Bokobza

et al. 2022

Preprint

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Genome engineering has become more accessible thanks to the RNA programmable endonucleases such as the CRISPR/Cas9 system. However, using this editing technology in synthetic organs called ‘organoids’ is still very inefficient. This is due to the delivery methods used for the CRISPR-Cas9 machinery, which include electroporation of CRISPR/Cas9 DNA, mRNA or ribonucleoproteins (RNPs) containing the CAS9-gRNA complex. However, these procedures are toxic to some extent for the organoids. Here we describe the use of the ‘Nanoblade’ technology, which outperformed by far knock-out (KO) levels achieved to date by gene editing in murine and human tissue derived organoids. We reached up to 80% of gene KO in organoids after treatment with nanoblades. Indeed, high-level nanoblade-mediated KO for the androgen receptor (AR) encoding gene and the cystic fibrosis transmembrane conductance regulator (CFTR) gene was achieved with single gRNA or dual gRNA containing nanoblades in murine prostate and colon organoids. Likewise, nanoblades achieved high levels of gene editing in human organoids ranging between 20% and 50%.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 KO in organoids and nanoblades simplify and allow rapid genome editing in organoids with little to no side-effects such as possible unwanted INDELS in off-target sites.

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A protocol for efficient CRISPR-Cas9-mediated knock-in in colorectal cancer patient-derived organoids

Cited by 12 publications

References 6 publications

Myoglobin in Brown Adipose Tissue: A Multifaceted Player in Thermogenesis

Myoglobin in Brown Adipose Tissue: A Multifaceted Player in Thermogenesis

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

Nanoblades allow high-level genome editing in organoids

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