Virus-Like Particle Mediated CRISPR/Cas9 Delivery for Efficient and Safe Genome Editing

Lyu, Pin; Wang, Luxi; Lu, Baisong

doi:10.3390/life10120366

Cited by 44 publications

(42 citation statements)

References 116 publications

(155 reference statements)

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“…[ 157 ] The remnant of the helical or icosahedral envelope and capsids of the virus in the VLP structure can efficiently encapsidate the genetic molecule within the virions of the host and deliver it to the targeted cells. [ 158 ] Viral infection or transfection, recombinant techniques, and cell‐free systems are the three main categories of viral capsid production. The first category describes a method for harvesting the empty capsids being fabricated as by‐products of the infected cells.…”

Section: Nanocarriersmentioning

confidence: 99%

Nanotechnology‐Assisted RNA Delivery: From Nucleic Acid Therapeutics to COVID‐19 Vaccines

et al. 2021

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In recent years, the main quest of science has been the pioneering of the groundbreaking biomedical strategies needed for achieving a personalized medicine. Ribonucleic acids (RNAs) are outstanding bioactive macromolecules identified as pivotal actors in regulating a wide range of biochemical pathways. The ability to intimately control the cell fate and tissue activities makes RNA‐based drugs the most fascinating family of bioactive agents. However, achieving a widespread application of RNA therapeutics in humans is still a challenging feat, due to both the instability of naked RNA and the presence of biological barriers aimed at hindering the entrance of RNA into cells. Recently, material scientists’ enormous efforts have led to the development of various classes of nanostructured carriers customized to overcome these limitations. This work systematically reviews the current advances in developing the next generation of drugs based on nanotechnology‐assisted RNA delivery. The features of the most used RNA molecules are presented, together with the development strategies and properties of nanostructured vehicles. Also provided is an in‐depth overview of various therapeutic applications of the presented systems, including coronavirus disease vaccines and the newest trends in the field. Lastly, emerging challenges and future perspectives for nanotechnology‐mediated RNA therapies are discussed.

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Section: Nanocarriersmentioning

confidence: 99%

Nanotechnology‐Assisted RNA Delivery: From Nucleic Acid Therapeutics to COVID‐19 Vaccines

et al. 2021

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“…An RNA segment of the mom gene of bacteriophage Mu (com) was strongly and specifically associated with a phage Com protein (COM) [120,121]. The com sequence inserted into the tetraloop of sgRNA had little or no influence on the package and activity of the Cas9-sgRNA RNP complex and interacted with the COM protein fused to the Gag polyprotein in the 3 -part of NC [122][123][124]. Besides Gag polyprotein, the G3C, V153L, and G177E gene-modified NEF protein (1100 copies per capsid) [125,126] and VPR protein (550 copies per capsid) [127,128] could be used as a carrier for the aptamer binding proteins [129].…”

Section: Delivery Of Cas9/sgrna Rnp With Virus-like Particlesmentioning

confidence: 99%

Points of View on the Tools for Genome/Gene Editing

Chuang

Lin

2021

IJMS

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Theoretically, a DNA sequence-specific recognition protein that can distinguish a DNA sequence equal to or more than 16 bp could be unique to mammalian genomes. Long-sequence-specific nucleases, such as naturally occurring Homing Endonucleases and artificially engineered ZFN, TALEN, and Cas9-sgRNA, have been developed and widely applied in genome editing. In contrast to other counterparts, which recognize DNA target sites by the protein moieties themselves, Cas9 uses a single-guide RNA (sgRNA) as a template for DNA target recognition. Due to the simplicity in designing and synthesizing a sgRNA for a target site, Cas9-sgRNA has become the most current tool for genome editing. Moreover, the RNA-guided DNA recognition activity of Cas9-sgRNA is independent of both of the nuclease activities of it on the complementary strand by the HNH domain and the non-complementary strand by the RuvC domain, and HNH nuclease activity null mutant (H840A) and RuvC nuclease activity null mutant (D10A) were identified. In accompaniment with the sgRNA, Cas9, Cas9(D10A), Cas9(H840A), and Cas9(D10A, H840A) can be used to achieve double strand breakage, complementary strand breakage, non-complementary strand breakage, and no breakage on-target site, respectively. Based on such unique characteristics, many engineered enzyme activities, such as DNA methylation, histone methylation, histone acetylation, cytidine deamination, adenine deamination, and primer-directed mutation, could be introduced within or around the target site. In order to prevent off-targeting by the lasting expression of Cas9 derivatives, a lot of transient expression methods, including the direct delivery of Cas9-sgRNA riboprotein, were developed. The issue of biosafety is indispensable in in vivo applications; Cas9-sgRNA packaged into virus-like particles or extracellular vesicles have been designed and some in vivo therapeutic trials have been reported.

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“…[35][36][37][38] Recently, we and others developed retrovirus-based MS2 bacteriophage (MS2) chimera for reversible delivery of the multi-component CRISPR-Cas9 technology into target cells. 16,[39][40][41] Redirecting the retroviral packaging machinery to respective components of MS2 allowed us to specifically deliver RNA species other than mRNA, including RNA polymerase III (RNA Pol III)-driven transcripts. 16 Similar to retroviruses, MS2 is a plus-and single-stranded RNA virus, whose genome is embedded within a capsid formed by the viral MS2 coat protein (MS2CP).…”

Section: Introductionmentioning

confidence: 99%

Improved alpharetrovirus-based Gag.MS2 particles for efficient and transient delivery of CRISPR-Cas9 into target cells

Baron¹,

Sens²,

Lange³

et al. 2022

Molecular Therapy - Nucleic Acids

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DNA-modifying technologies, such as the CRISPR-Cas9 system, are promising tools in the field of gene and cell therapies. However, high and prolonged expression of DNA-modifying enzymes may cause cytotoxic and genotoxic side effects and is therefore unwanted in therapeutic approaches. Consequently, development of new and potent short-term delivery methods is of utmost importance. Recently, we developed non-integrating gammaretrovirus-and MS2 bacteriophage-based Gag.MS2 (g.Gag.MS2) particles for transient transfer of nonretroviral CRISPR-Cas9 RNA into target cells. In the present study, we further improved the technique by transferring the system to the alpharetroviral vector platform (a.Gag.MS2), which significantly increased CRISPR-Cas9 delivery into target cells and allowed efficient targeted knockout of endogenous TP53/Trp53 genes in primary murine fibroblasts as well as primary human fibroblasts, hepatocytes, and cord-blood-derived CD34 + stem and progenitor cells. Strikingly, co-packaging of Cas9 mRNA and multiple single guide RNAs (sgRNAs) into a.Gag.MS2 chimera displayed efficient targeted knockout of up to three genes. Co-transfection of single-stranded DNA donor oligonucleotides during CRISPR-Cas9 particle production generated all-in-one particles, which mediated up to 12.5% of homology-directed repair in primary cell cultures. In summary, optimized a.Gag.MS2 particles represent a versatile tool for short-term delivery of DNA-modifying enzymes into a variety of target cells, including primary murine and human cells.

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Virus-Like Particle Mediated CRISPR/Cas9 Delivery for Efficient and Safe Genome Editing

Cited by 44 publications

References 116 publications

Nanotechnology‐Assisted RNA Delivery: From Nucleic Acid Therapeutics to COVID‐19 Vaccines

Nanotechnology‐Assisted RNA Delivery: From Nucleic Acid Therapeutics to COVID‐19 Vaccines

Points of View on the Tools for Genome/Gene Editing

Improved alpharetrovirus-based Gag.MS2 particles for efficient and transient delivery of CRISPR-Cas9 into target cells

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