Cationic lipopolymeric nanoplexes containing the CRISPR/Cas9 ribonucleoprotein for genome surgery

Sahel, Deepak Kumar; Salman, Mohammad; Azhar, Mohd; Goswami, Sangam Giri; Singh, Vivek; Dalela, Manu; Mohanty, Sujata; Mittal, Anupama; Ramalingam, Sivaprakash

doi:10.1039/d2tb00645f

Cited by 7 publications

(22 citation statements)

References 40 publications

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“…The increase in particle size of pCas9-loaded lipopolymeric nanoplexes w.r.t to the blank lipopolymeric nanoplexes is also an indication of complex formation and this kind of observation has been already reported in the literature. [5] The morphological structure of the pCas9-loaded lipopolymeric nanoplexes is shown in Figure 3c, which was found to be similar to the previously reported literature. [16] However, the blank lipopolymeric nanoplexes and the pCas9-loaded lipopolymeric nanoplexes showed a zeta potential of 16.6 ± 1.6 mV and 5.9 ± 0.5, respectively (Figure 3d).…”

Section: Formulation Development and Characterizationsupporting

confidence: 87%

“…A previously reported method was adopted for the preparation of pCas9‐loaded lipopolymeric nanoplexes. [ 5 ] Briefly, 10 mg of mPEG b‐(CB‐g‐cationic chain; g‐Chol; g‐Morph) polymer was accurately weighed and dissolved in 600 µL of dichloromethane (DCM). Further, 100 µL of nuclease‐free water (NFW) was added to the polymer solution, followed by pipetting to get a primary emulsion (w/o).…”

Section: Methodsmentioning

confidence: 99%

“…The synthesis, characterization and structural composition of mPEG-b-(CB-g-cationic chain; g-Chol; g-Morph) polymer were similar to our previous work and can be accessed from reference. [5,10] The final structure of the mPEG-b-(CB-g-cationic chain; g-Chol; g-Morph) is shown in Figure 1a.…”

Section: Synthesis Of Cationic Lipopolymer Mpeg-b-(cb-{g-cationic Cha...mentioning

confidence: 99%

“…The previously reported method with slight modification was adopted for the transfection experiment. [5,12] Briefly, ARPE-19 cells were seeded in a 24-well cell culture plate with a density of 2 × 10 4 cells/well and allowed to grow overnight at 37 °C with 5% CO 2. Next day, the media was replaced by 250 μL OptiMEM media (ThermoFisher, MA, USA) containing pCas9-TURBO-GFPloaded lipopolymeric nanoplexes (pCas9: blank lipopolymeric nanoplexes; 1:20) with a net 1 ug of the pCas9-TURBO-GFP plasmid and cells were incubated at 37 °C with 5% CO 2 .…”

Section: Transfection Assay In Mammalian Cellsmentioning

confidence: 99%

“…[ 2 ] The in vitro and in vivo delivery of all three forms of CRISPR is quite challenging owing to the high molecular weight, supranegative charge, hydrophilicity, degradation in the extracellular environment by nucleases, etc. [ 5 ] However, the viral vectors have been utilized for the delivery of CRISPR components, specifically the plasmid, but possess ample limitations such as immunogenicity, limited payload capacity, mutagenesis, etc. [ 6 ] For instance, the payload capacity of the adeno‐associated viruses (AVVs) vector is ≈4.7 kb, however, the Cas9 gene itself has a size of ≈4 kb.…”

Section: Introductionmentioning

confidence: 99%

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Lipopolymeric Nanocarrier Enables Effective Delivery of CRISPR/Cas9 Expressing Plasmid

Sahel

Goswami

Jatyan

et al. 2023

Macromol. Rapid Commun.

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CRISPR/Cas9 has proven its accuracy and precision for gene editing by making a double-strand break at the predetermined site. Despite being a mainstream gene editing tool, CRISPR/Cas9 has limitations for its in vivo delivery due to the physico-chemical properties such as high molecular weight, supranegative charge, degradation in the presence of nucleases, etc. Hereby, a cationic lipopolymer is explored for its efficiency in delivering CRISPR/Cas9 plasmid (pCas9) in vitro and in vivo. The lipopolymer is utilized to form blank cationic nanoplexes having a zeta potential of +15.8 ± 0.7 mV. Being cationic, the blank nanoplexes are able to condense the pCas9 plasmid at a ratio of 1:20 with a complexation efficiency of ≈98% and show a size and zeta potential of ≈141 ± 16 nm and 4.2 mV ± 0.7, respectively. The pCas9-loaded nanoplexes show a transfection efficiency of ≈69% in ARPE-19 cells and show ≈22% of indel frequency, indicating the successful translation of Cas9 protein and guide RNA in the cytosol. Further, they are found to be stable under in vivo environment when given intravenously in Swiss albino mice. These lipopolymeric nanoplexes can be a potential carrier for CRISPR plasmids for genome editing applications.

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Section: Formulation Development and Characterizationsupporting

confidence: 87%

Section: Methodsmentioning

confidence: 99%

Section: Synthesis Of Cationic Lipopolymer Mpeg-b-(cb-{g-cationic Cha...mentioning

confidence: 99%

Section: Transfection Assay In Mammalian Cellsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Lipopolymeric Nanocarrier Enables Effective Delivery of CRISPR/Cas9 Expressing Plasmid

Sahel

Goswami

Jatyan

et al. 2023

Macromol. Rapid Commun.

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CRISPR/Cas9 Genome Editing for Tissue‐Specific In Vivo Targeting: Nanomaterials and Translational Perspective

et al. 2023

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Clustered randomly interspaced short palindromic repeats (CRISPRs) and its associated endonuclease protein, i.e., Cas9, have been discovered as an immune system in bacteria and archaea; nevertheless, they are now being adopted as mainstream biotechnological/molecular scissors that can modulate ample genetic and nongenetic diseases via insertion/deletion, epigenome editing, messenger RNA editing, CRISPR interference, etc. Many Food and Drug Administration‐approved and ongoing clinical trials on CRISPR adopt ex vivo strategies, wherein the gene editing is performed ex vivo, followed by reimplantation to the patients. However, the in vivo delivery of the CRISPR components is still under preclinical surveillance. This review has summarized the nonviral nanodelivery strategies for gene editing using CRISPR/Cas9 and its recent advancements, strategic points of view, challenges, and future aspects for tissue‐specific in vivo delivery of CRISPR/Cas9 components using nanomaterials.

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Potent anti-inflammatory and anti-apoptotic activities of electrostatically complexed C-peptide nanospheres ameliorate diabetic nephropathy

Singh,

Salunkhe,

Chitkara

et al. 2024

Biomaterials Advances

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Cationic lipopolymeric nanoplexes containing the CRISPR/Cas9 ribonucleoprotein for genome surgery

Abstract: sgRNA/Cas9 ribonucleoproteins (RNPs) provide a site-specific robust gene-editing approach avoiding the mutagenesis and unwanted off-target effects. However, the high molecular weight (~165 kDa), hydrophilicity and net supranegative charge (~ -20...

Cited by 7 publications

References 40 publications

Lipopolymeric Nanocarrier Enables Effective Delivery of CRISPR/Cas9 Expressing Plasmid

Lipopolymeric Nanocarrier Enables Effective Delivery of CRISPR/Cas9 Expressing Plasmid

CRISPR/Cas9 Genome Editing for Tissue‐Specific In Vivo Targeting: Nanomaterials and Translational Perspective

Potent anti-inflammatory and anti-apoptotic activities of electrostatically complexed C-peptide nanospheres ameliorate diabetic nephropathy

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