Robust genome editing in adult vascular endothelium by nanoparticle delivery of CRISPR-Cas9 plasmid DNA

Introduction: Therapeutic delivery of CRISPR system components to induce in vivo genome editing in postnatal and adult life has great translational potential. Recent studies employing non-viral delivery of small guide RNA (gRNA) and Cas9 mRNA have achieved efficient genome editing in adult mice. However, as often seen in other RNA therapeutic studies with non-viral delivery of antisense and siRNA, the efficiency is limited to the liver. Hypothesis: Novel nanoparticle can therapeutically deliver the CRISPR system to selectively target cardiovascular endothelium in adult mice. Methods: We developed novel PLGA-based nanoparticles which was for the first time shown to be uptaken efficiently by the vascular endothelium without specific liver accumulation following i.v. administration. Mixture of the nanoparticle:plasmid DNA expressing Cas9 under the control of the human CDH5 promoter (EC-specific) and gRNA by the U6 promoter was administered i.v. to adult mice. Seven to ten days later, various organ tissues were collected for analysis of the efficiency of genomic editing and knockout of protein expression. The phenotype of CRISPR-mediated in vivo knockout of Pik3cg which encodes the G protein-coupled receptor-activated p110gamma isoform of PI3K was compared to Pik3cg null mice in response to sepsis challenge. Results: Therapeutic delivery of nanoparticles loaded with the all-in-one CRISPR plasmid DNA induced highly efficient genome editing in endothelial cells (ECs) of the cardiovascular system including heart, lung, and aorta in adult mice. The Indel rate was as great as 50% in ECs isolated from these vascular beds. Immunostaining and Western blotting demonstrated greater than 70% decrease of protein expression in ECs. Pik3cg -gRNA-induced genome editing diminished p110γPI3K expression in pulmonary vascular ECs, which led to impaired vascular repair and resolution of inflammation after sepsis challenge as seen in Pik3cg -/- mice. Conclusion: We have developed a simple and highly efficient method for in vivo genome editing selectively targeting the vascular endothelium. This strategy will greatly facilitate cardiovascular research and may enable therapeutic genome editing for prevention and treatment of cardiovascular diseases.

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Versatile Redox-Responsive Polyplexes for the Delivery of Plasmid DNA, Messenger RNA, and CRISPR-Cas9 Genome-Editing Machinery

ACS Applied Materials & Interfaces ◽

10.1021/acsami.8b09642 ◽

2018 ◽

Vol 10 (38) ◽

pp. 31915-31927 ◽

Cited By ~ 12

Author(s):

Yuyuan Wang ◽

Ben Ma ◽

Amr A. Abdeen ◽

Guojun Chen ◽

Ruosen Xie ◽

...

Keyword(s):

Genome Editing ◽

Plasmid Dna ◽

Messenger Rna ◽

Redox Responsive

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Highly efficient and specific genome editing in human cells with paired CRISPR-Cas9 nickase ribonucleoproteins

10.1101/766493 ◽

2019 ◽

Author(s):

Jacob Lamberth ◽

Laura Daley ◽

Pachai Natarajan ◽

Stanislav Khoruzhenko ◽

Nurit Becker ◽

...

Keyword(s):

Cell Culture ◽

Genome Editing ◽

Plasmid Dna ◽

High Efficiency ◽

High Specificity ◽

Human Cells ◽

Dna And Rna ◽

Genome Modification ◽

Delivery Technology ◽

Target Effects

ABSTRACTCRISPR technology has opened up many diverse genome editing possibilities in human somatic cells, but has been limited in the therapeutic realm by both potential off-target effects and low genome modification efficiencies. Recent advancements to combat these limitations include delivering Cas9 nucleases directly to cells as highly purified ribonucleoproteins (RNPs) instead of the conventional plasmid DNA and RNA-based approaches. Here, we extend RNP-based delivery in cell culture to a less characterized CRISPR format which implements paired Cas9 nickases. The use of paired nickase Cas9 RNP system, combined with a GMP-compliant non-viral delivery technology, enables editing in human cells with high specificity and high efficiency, a development that opens up the technology for further exploration into a more therapeutic role.

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292. In Vivo Nanoparticle Delivery for Site-Specific Genome Editing of Hematopoietic Cells in a Humanized Mouse

Molecular Therapy ◽

10.1016/s1525-0016(16)36096-8 ◽

2012 ◽

Vol 20 ◽

pp. S115

Keyword(s):

Genome Editing ◽

Hematopoietic Cells ◽

Humanized Mouse ◽

Site Specific ◽

Nanoparticle Delivery

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Advanced microinjection protocol for gene manipulation using the model newt Pleurodeles waltl

The International Journal of Developmental Biology ◽

10.1387/ijdb.180297th ◽

2019 ◽

Vol 63 (6-7) ◽

pp. 281-286 ◽

Cited By ~ 1

Author(s):

Toshinori Hayashi ◽

Mie Nakajima ◽

Mitsuki Kyakuno ◽

Kanako Doi ◽

Ikumi Manabe ◽

...

Keyword(s):

Developmental Biology ◽

Genome Editing ◽

Plasmid Dna ◽

Experimental System ◽

Gene Manipulation ◽

Experimental Animals ◽

The Core ◽

Research Fields ◽

Injection Protocol ◽

Pleurodeles Waltl

Urodele amphibian newts have an outstanding history as experimental animals in various research fields such as developmental biology and regeneration biology. We have reported a model experimental system using the Spanish newt, Pleurodeles waltl, and it enables reverse/molecular genetics through gene manipulation. Microinjection is one of the core techniques in gene manipulation in newts. In the present study, we examined the conditions of the microinjection method, such as egg preparation, de-jelly solution, and formulation of injection medium. We have successfully optimized the injection protocol for P. waltl newts, and our improved protocol is more efficient and lower in cost than previous methods. This protocol can be used for the microinjection of plasmid DNA with I-SceI or mRNA, as well as genome editing using the CRISPR-Cas9 system. This protocol will facilitate research through gene manipulation in newts.

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One-plasmid based CRISPR-Cas9 editing for gene deletion in Lactococcus lactis

10.21203/rs.3.rs-306012/v1 ◽

2021 ◽

Author(s):

José Miguel Miquelão Santos ◽

Gabriel António Amaro Monteiro ◽

Duarte Miguel de França Teixeira dos Prazeres ◽

Sofia de Oliveira Dias Duarte

Keyword(s):

Genome Editing ◽

Lactococcus Lactis ◽

Plasmid Dna ◽

Genome Engineering ◽

Gene Deletion ◽

Deletion Mutants ◽

Cell Factories ◽

Yield And Quality ◽

Delivery Vehicles

Abstract Lactococcus lactis strains are promising cell factories and delivery vehicles of plasmid DNA and recombinant protein for therapeutic applications. However, the limited yields of recombinant molecules obtained with these bacteria limits their wide applicability. Genome engineering of this host may solve the problem. However, the current genome editing toolbox available for L. lactis is either too laborious or incapable of large edits, limiting the scope of strain editing experiments. In this work, the basis for a one-plasmid CRISPR-Cas9 based genome editing plasmid was developed and tested. The new plasmid (pTCas9dO) adapted from the pKCcas9dO plasmid was used to delete 657 bp of the lactococcal nuclease nth of L. lactis subsp. lactis LMG19460, with the aim of improving yield and quality of plasmid DNA replicated in this strain. Although deletion mutants were successfully generated, plasmid curing was unsuccessful. Thus, further modifications are required before the plasmid is truly applicable for genome editing experiments. Unexpectedly, the generated deletion mutants generated a roughly 40% decrease in plasmid yield alongside with a decrease in the quality of produced pDNA.

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Reducible Branched Ester-Amine Quadpolymers (rBEAQs) Codelivering Plasmid DNA and RNA Oligonucleotides Enable CRISPR/Cas9 Genome Editing

ACS Applied Materials & Interfaces ◽

10.1021/acsami.8b20206 ◽

2019 ◽

Vol 11 (11) ◽

pp. 10472-10480 ◽

Cited By ~ 13

Author(s):

Yuan Rui ◽

David R. Wilson ◽

Katie Sanders ◽

Jordan J. Green

Keyword(s):

Genome Editing ◽

Plasmid Dna ◽

Dna And Rna

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Systemic nanoparticle delivery of CRISPR-Cas9 ribonucleoproteins for effective tissue specific genome editing

Nature Communications ◽

10.1038/s41467-020-17029-3 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 15

Author(s):

Tuo Wei ◽

Qiang Cheng ◽

Yi-Li Min ◽

Eric N. Olson ◽

Daniel J. Siegwart

Keyword(s):

Genome Editing ◽

Tissue Specific ◽

Nanoparticle Delivery

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CRISPR/Cas9 Delivery System Engineering for Genome Editing in Therapeutic Applications

Pharmaceutics ◽

10.3390/pharmaceutics13101649 ◽

2021 ◽

Vol 13 (10) ◽

pp. 1649

Author(s):

Hao Cheng ◽

Feng Zhang ◽

Yang Ding

Keyword(s):

Genome Editing ◽

Plasmid Dna ◽

Biomedical Applications ◽

Viral Vectors ◽

Recent Progress ◽

Disease Modeling ◽

Gene Correction ◽

Cas9 Protein ◽

Clinical Potential ◽

Delivery Technology

The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) systems have emerged as a robust and versatile genome editing platform for gene correction, transcriptional regulation, disease modeling, and nucleic acids imaging. However, the insufficient transfection and off-target risks have seriously hampered the potential biomedical applications of CRISPR/Cas9 technology. Herein, we review the recent progress towards CRISPR/Cas9 system delivery based on viral and non-viral vectors. We summarize the CRISPR/Cas9-inspired clinical trials and analyze the CRISPR/Cas9 delivery technology applied in the trials. The rational-designed non-viral vectors for delivering three typical forms of CRISPR/Cas9 system, including plasmid DNA (pDNA), mRNA, and ribonucleoprotein (RNP, Cas9 protein complexed with gRNA) were highlighted in this review. The vector-derived strategies to tackle the off-target concerns were further discussed. Moreover, we consider the challenges and prospects to realize the clinical potential of CRISPR/Cas9-based genome editing.

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