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Engineering Virus-like Particles for the Delivery of Genome Editing Enzymes.

Record Type:	Language materials, printed : Monograph/item
Title/Author:	Engineering Virus-like Particles for the Delivery of Genome Editing Enzymes./
Author:	Rousseau, Beth.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2022,
Description:	173 p.
Notes:	Source: Dissertations Abstracts International, Volume: 84-01, Section: B.
Contained By:	Dissertations Abstracts International84-01B.
Subject:	Bioengineering. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=29274875
ISBN:	9798438777410

Engineering Virus-like Particles for the Delivery of Genome Editing Enzymes.
Rousseau, Beth.

Engineering Virus-like Particles for the Delivery of Genome Editing Enzymes. - Ann Arbor : ProQuest Dissertations & Theses, 2022 - 173 p.

Source: Dissertations Abstracts International, Volume: 84-01, Section: B.

Thesis (Ph.D.)--University of Michigan, 2022.

This item must not be sold to any third party vendors.

Genome editing with Cas9 is a powerful method of investigating the roles of genes in biology. S. pyogenes Cas9 catalyzes a precise, blunt, double-stranded break in DNA when directed toward a genomic locus complementary to a programmable guide RNA and adjacent to the sequence 5’-NGG-3’. However, prolonged expression of the Cas9 ribonucleoprotein (RNP) in cells can increase off-target cleavage events. Transient Cas9 RNP transduction can mitigate the risk of off-target events by reducing both the length of time that cells are exposed to Cas9 RNPs and controlling the dose of RNP administered. Common methods of delivering Cas9 RNPs include electroporation, lipid-based transfections, nanoparticles, and virus-like particles (VLPs). VLPs are particles similar in form and function to a virus but lacking a viral genome. We chose to construct VLPs for Cas9 delivery because they require no specialized equipment to transduce cells, are inexpensive, can be scaled up easily, have relatively low toxicity, and can be pseudotyped with different viral envelope proteins which can enable delivery to a wide range of cells. VLPs derived from the Murine Leukemia Virus (MLV) are known to serve as a vehicle for the efficient transduction of proteins, including Cas9 and other genome editing enzymes. Gag and GagPol are the two polyproteins that comprise the interior of an MLV virion. Gag expressed independently of other viral transcripts and proteins spontaneously forms a VLP. A protein fused to the C-terminus of Gag is loaded into MLV VLPs concomitantly with their creation. In an effort to improve VLP efficacy, we have made novel Moloney MLV VLPs with codon optimized Gag. Codon optimization improves VLP titer by a factor of ten. Another optimization that we have made to VLPs is the elimination of functional reverse transcriptase and integrase domains from GagPol. Although these domains are important for the lifecycle of the virus, we have found them to be inessential for Cas9 delivery by VLP. We have also found that VLPs can deliver two enzymatically active cargo proteins at once, Cre and Cas9. Finally, we show that delivery of prime editing enzymes can be achieved with VLPs.

ISBN: 9798438777410Subjects--Topical Terms:

598252
Bioengineering.
Subjects--Index Terms:

Genome editing

Engineering Virus-like Particles for the Delivery of Genome Editing Enzymes.
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520 $a Genome editing with Cas9 is a powerful method of investigating the roles of genes in biology. S. pyogenes Cas9 catalyzes a precise, blunt, double-stranded break in DNA when directed toward a genomic locus complementary to a programmable guide RNA and adjacent to the sequence 5’-NGG-3’. However, prolonged expression of the Cas9 ribonucleoprotein (RNP) in cells can increase off-target cleavage events. Transient Cas9 RNP transduction can mitigate the risk of off-target events by reducing both the length of time that cells are exposed to Cas9 RNPs and controlling the dose of RNP administered. Common methods of delivering Cas9 RNPs include electroporation, lipid-based transfections, nanoparticles, and virus-like particles (VLPs). VLPs are particles similar in form and function to a virus but lacking a viral genome. We chose to construct VLPs for Cas9 delivery because they require no specialized equipment to transduce cells, are inexpensive, can be scaled up easily, have relatively low toxicity, and can be pseudotyped with different viral envelope proteins which can enable delivery to a wide range of cells. VLPs derived from the Murine Leukemia Virus (MLV) are known to serve as a vehicle for the efficient transduction of proteins, including Cas9 and other genome editing enzymes. Gag and GagPol are the two polyproteins that comprise the interior of an MLV virion. Gag expressed independently of other viral transcripts and proteins spontaneously forms a VLP. A protein fused to the C-terminus of Gag is loaded into MLV VLPs concomitantly with their creation. In an effort to improve VLP efficacy, we have made novel Moloney MLV VLPs with codon optimized Gag. Codon optimization improves VLP titer by a factor of ten. Another optimization that we have made to VLPs is the elimination of functional reverse transcriptase and integrase domains from GagPol. Although these domains are important for the lifecycle of the virus, we have found them to be inessential for Cas9 delivery by VLP. We have also found that VLPs can deliver two enzymatically active cargo proteins at once, Cre and Cas9. Finally, we show that delivery of prime editing enzymes can be achieved with VLPs.
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