Direct Cytosolic Delivery of CRISPR/Cas9-Ribonucleoprotein for Efficient Gene Editing

Mout, Rubul; Ray, Moumita; Tonga, Gülen Yesilbag; Lee, Yi-Wei; Tay, Tristan; Sasaki, Kyoyu; Rotello, Vincent M.

doi:10.1021/acsnano.6b07600

Cited by 437 publications

(385 citation statements)

References 30 publications

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“…Any punctate distribution (see Figure 2B) of labelled-Cas9En should be avoided from counting as direct cytoplasmic and nuclear delivery. Please see Mout et al ., 2017 for more details.…”

Section: Discussionmentioning

confidence: 99%

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Cytosolic and Nuclear Delivery of CRISPR/Cas9-ribonucleoprotein for Gene Editing Using Arginine Functionalized Gold Nanoparticles

Mout

Rotello

2017

BIO-PROTOCOL

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In this protocol, engineered Cas9-ribonucleoprotein (Cas9 protein and sgRNA, together called Cas9-RNP) and gold nanoparticles are used to make nanoassemblies that are employed to deliver Cas9-RNP into cell cytoplasm and nucleus. Cas9 protein is engineered with an N-terminus glutamic acid tag (E-tag or En, where n = the number of glutamic acid in an E-tag and usually n = 15 or 20), C-terminus nuclear localizing signal (NLS), and a C-terminus 6xHis-tag. [Cas9En hereafter] To use this protocol, the first step is to generate the required materials (gold nanoparticles, recombinant Cas9En, and sgRNA). Laboratory-synthesis of gold nanoparticles can take up to a few weeks, but can be synthesized in large batches that can be used for many years without compromising the quality. Cas9En can be cloned from a regular SpCas9 gene (Addgene plasmid id = 47327), and expressed and purified using standard laboratory procedures which are not a part of this protocol. Similarly, sgRNA can be laboratory-synthesized using in vitro transcription from a template gene (Addgene plasmid id = 51765) or can be purchased from various sources. Once these materials are ready, it takes about ~30 min to make the Cas9En-RNP complex and 10 min to make the Cas9En-RNP/nanoparticles nanoassemblies, which are immediately used for delivery (Figure 1). Complete delivery (90–95% cytoplasmic and nuclear delivery) is achieved in less than 3 h. Follow-up editing experiments require additional time based on users’ need. Synthesis of arginine functionalized gold nanoparticles (ArgNPs) (Yang et al., 2011), expression of recombinant Cas9En, and in vitro synthesis of sgRNA is reported elsewhere (Mout et al., 2017). We report here only the generation of the delivery vehicle i.e., the fabrication of Cas9En-RNP/ArgNPs nanoassembly.

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“…Any punctate distribution (see Figure 2B) of labelled-Cas9En should be avoided from counting as direct cytoplasmic and nuclear delivery. Please see Mout et al ., 2017 for more details.…”

Section: Discussionmentioning

confidence: 99%

“…Synthesis of arginine functionalized gold nanoparticles (ArgNPs) (Yang et al ., 2011), expression of recombinant Cas9En, and in vitro synthesis of sgRNA is reported elsewhere (Mout et al ., 2017). We report here only the generation of the delivery vehicle i.e.…”

mentioning

confidence: 99%

Cytosolic and Nuclear Delivery of CRISPR/Cas9-ribonucleoprotein for Gene Editing Using Arginine Functionalized Gold Nanoparticles

Mout

Rotello

2017

BIO-PROTOCOL

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“…Gold nanoparticles are co-assembled with the Cas9 protein engineered with a glutamate peptide tag and sgRNA into nanoparticles. This nanoparticle-mediated delivery system achieves greater than 90% delivery efficiency and 30% gene editing efficiency in a wide variety of cell types 82 . The nanoparticle-mediated delivery of the engineered Cas9 protein and sgRNA is achieved through a cholesterol-dependent membrane fusion process that is distinct from cellular endocytosis, which may underline the remarkable delivery efficiency of this system 82 .…”

Section: Physical and Non-viral Delivery Of Crispr-cas9mentioning

confidence: 99%

Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications

Liu

zhang

Líu

et al. 2017

Journal of Controlled Release

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The CRISPR-Cas9 genome-editing system is a part of the adaptive immune system in archaea and bacteria to defend against invasive nucleic acids from phages and plasmids. The single guide RNA (sgRNA) of the system recognizes its target sequence in the genome, and the Cas9 nuclease of the system acts as a pair of scissors to cleave the double strands of DNA. Since its discovery, CRISPR-Cas9 has become the most robust platform for genome engineering in eukaryotic cells. Recently, the CRISPR-Cas9 system has triggered enormous interest in therapeutic applications. CRISPR-Cas9 can be applied to correct disease-causing gene mutations or engineer T cells for cancer immunotherapy. The first clinical trial using the CRISPR-Cas9 technology was conducted in 2016. Despite the great promise of the CRISPR-Cas9 technology, several challenges remain to be tackled before its successful applications for human patients. The greatest challenge is the safe and efficient delivery of the CRISPR-Cas9 genome-editing system to target cells in human body. In this review, we will introduce the molecular mechanism and different strategies to edit genes using the CRISPR-Cas9 system. We will then highlight the current systems that have been developed to deliver CRISPR-Cas9 in vitro and in vivo for various therapeutic purposes.

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“…31 Thus, packaging these elements through supramolecular chemistry may be a major limitation for designing delivery vehicles. Moreover, incorporation of additional spare DNA (of size in kbp) for multiple applications may further complicate the vector design.…”

Section: Challengesmentioning

confidence: 99%

In Vivo Delivery of CRISPR/Cas9 for Therapeutic Gene Editing: Progress and Challenges

et al. 2017

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Direct Cytosolic Delivery of CRISPR/Cas9-Ribonucleoprotein for Efficient Gene Editing

Cited by 437 publications

References 30 publications

Cytosolic and Nuclear Delivery of CRISPR/Cas9-ribonucleoprotein for Gene Editing Using Arginine Functionalized Gold Nanoparticles

Cytosolic and Nuclear Delivery of CRISPR/Cas9-ribonucleoprotein for Gene Editing Using Arginine Functionalized Gold Nanoparticles

Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications

In Vivo Delivery of CRISPR/Cas9 for Therapeutic Gene Editing: Progress and Challenges

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