Lipopeptide‐Based Nanosome‐Mediated Delivery of Hyperaccurate CRISPR/Cas9 Ribonucleoprotein for Gene Editing

Thach, Trung Thanh; Bae, Do Hyun; Kim, Nam Hyeong; Kang, Eun Sung; Lee, Bok Soo; Han, Kayoung; Kwak, Myung-Jun; Choi, Hojae; Nam, Ji Young; Bae, Taegeun; Suh, Minah; Hur, Junho K.

doi:10.1002/smll.201903172

Cited by 10 publications

(5 citation statements)

References 29 publications

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“…However, long-term exposure of foreign DNA fragments and associated overexpression of functional Cas9sg may consequently increase the risk of insertional mutagenesis [14] as well as off-target effects [15]. To overcome these obstacles, cytosolic delivery of Cas9sg is a promising alternative with therapeutic potentials [16][17][18][19][20][21][22]. Although the carrier-dependent delivery can effectively transport Cas9sg into cells, the biosafety of carriers and complicated preparation process to assemble the necessary delivery platform are obstacles stifling biomedical applications.…”

Section: Introductionmentioning

confidence: 99%

Carrier-Free Cellular Transport of CRISPR/Cas9 Ribonucleoprotein for Genome Editing by Cold Atmospheric Plasma

Cui

Jiang

Zhou

et al. 2021

Biology

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A carrier-free CRISPR/Cas9 ribonucleoprotein delivery strategy for genome editing mediated by a cold atmospheric plasma (CAP) is described. The CAP is promising in many biomedical applications due to efficient production of bioactive ionized species. The MCF-7 cancer cells after CAP exposure exhibit increased extracellular reactive oxygen and nitrogen species (RONS) and altered membrane potential and permeability. Hence, transmembrane transport of Ca2+ into the cells increases and accelerates ATP hydrolysis, resulting in enhanced ATP-dependent endocytosis. Afterwards, the increased Ca2+ and ATP contents promote the release of cargo into cytoplasm due to the enhanced endosomal escape. The increased membrane permeability also facilitates passive diffusion of foreign species across the membrane into the cytosol. After CAP exposure, the MCF-7 cells incubated with Cas9 ribonucleoprotein (Cas9-sgRNA complex, Cas9sg) with a size of about 15 nm show 88.9% uptake efficiency and 65.9% nuclear import efficiency via passive diffusion and ATP-dependent endocytosis pathways. The efficient transportation of active Cas9sg after the CAP treatment leads to 21.7% and 30.2% indel efficiencies in HEK293T and MCF-7 cells, respectively. This CAP-mediated transportation process provides a simple and robust alternative for the delivery of active CRISPR/Cas9 ribonucleoprotein. Additionally, the technique can be extended to other macro-biomolecules and nanomaterials to cater to different biomedical applications.

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Section: Introductionmentioning

confidence: 99%

Carrier-Free Cellular Transport of CRISPR/Cas9 Ribonucleoprotein for Genome Editing by Cold Atmospheric Plasma

Cui

Jiang

Zhou

et al. 2021

Biology

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“…PT 24 showed comparable efficiency with Lipofectamine 2000 in editing the HPRT1 gene in several cell lines. Similarly, a blood-brain barrier permeable peptide dNP2 was conjugated with three different saturated fatty acids including caprylic acid (C8), decanoic acid (C10) and myristic acid (C14) to yield lipopeptides for Cas9 RNP delivery 189 . HypaCas9 is a hyper-accurate SpCas9 with improved targeting accuracy produced by targeted mutagenesis within the REC3 domain 190 .…”

Section: Materials For Rnp Deliverymentioning

confidence: 99%

“…HypaCas9 is a hyper-accurate SpCas9 with improved targeting accuracy produced by targeted mutagenesis within the REC3 domain 190 . The caprylic acid-modified peptide C8dNP2 exhibited the highest ability to form homologous nanosomes, and efficiently delivered HypaCas9 RNP into HEK and GBM cells with efficiencies higher than Lipofectamine 2000 and CRISPR MAX 189 . In another study, modification of lipopeptides with targeting peptides enabled cell-selective gene editing 191 .…”

Section: Materials For Rnp Deliverymentioning

confidence: 99%

Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing

Song¹,

Shen²,

Li³

et al. 2021

Theranostics

217

158

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CRISPR/Cas9 genome editing has gained rapidly increasing attentions in recent years, however, the translation of this biotechnology into therapy has been hindered by efficient delivery of CRISPR/Cas9 materials into target cells. Direct delivery of CRISPR/Cas9 system as a ribonucleoprotein (RNP) complex consisting of Cas9 protein and single guide RNA (sgRNA) has emerged as a powerful and widespread method for genome editing due to its advantages of transient genome editing and reduced off-target effects. In this review, we summarized the current Cas9 RNP delivery systems including physical approaches and synthetic carriers. The mechanisms and beneficial roles of these strategies in intracellular Cas9 RNP delivery were reviewed. Examples in the development of stimuli-responsive and targeted carriers for RNP delivery are highlighted. Finally, the challenges of current Cas9 RNP delivery systems and perspectives in rational design of next generation materials for this promising field will be discussed.

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“…The use of encapsulated HypaRNP in an in vivo model resulted in rapid nuclear localization without causing cytotoxicity. Thus, the HypaRNP effectively silenced endogenous eGFP in human embryonic kidney cells and glioblastoma cells ( Saadati and Hosseini, 2012 ; Thach et al, 2019 ). This strategy was also used to modify the dipeptidyl peptidase-4 gene (DPP-4) to upregulate the glucagon-like peptide 1 in a murine model of type 2 diabetes mellitus (T2DM) db/db mice.…”

Section: Non-viral Nano Delivery Systemsmentioning

confidence: 99%

Nanocarriers: A novel strategy for the delivery of CRISPR/Cas systems

et al. 2022

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In recent decades, clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) has become one of the most promising genome-editing tools for therapeutic purposes in biomedical and medical applications. Although the CRISPR/Cas system has truly revolutionized the era of genome editing, the safe and effective delivery of CRISPR/Cas systems represents a substantial challenge that must be tackled to enable the next generation of genetic therapies. In addition, there are some challenges in the in vivo delivery to the targeted cells/tissues. Nanotechnology-based drug delivery systems can be employed to overcome this issue. This review discusses different types and forms of CRISPR/Cas systems and the current CRISPR/Cas delivery systems, including non-viral carriers such as liposomes, polymeric, and gold particles. The focus then turns to the viral nanocarriers which have been recently used as a nanocarrier for CRISPR/Cas delivery.

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Lipopeptide‐Based Nanosome‐Mediated Delivery of Hyperaccurate CRISPR/Cas9 Ribonucleoprotein for Gene Editing

Cited by 10 publications

References 29 publications

Carrier-Free Cellular Transport of CRISPR/Cas9 Ribonucleoprotein for Genome Editing by Cold Atmospheric Plasma

Carrier-Free Cellular Transport of CRISPR/Cas9 Ribonucleoprotein for Genome Editing by Cold Atmospheric Plasma

Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing

Nanocarriers: A novel strategy for the delivery of CRISPR/Cas systems

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