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

Record Type:	Electronic resources : Monograph/item
Title/Author:	Engineering Virus-like Particles for the Delivery of Genome Editing Enzymes./
Author:	Rousseau, Beth.
Description:	1 online resource (173 pages)
Notes:	Source: Dissertations Abstracts International, Volume: 84-01, Section: B.
Contained By:	Dissertations Abstracts International84-01B.
Subject:	Biochemistry. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=29274875click for full text (PQDT)
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. - 1 online resource (173 pages)

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

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

Includes bibliographical references

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.

Electronic reproduction.
Ann Arbor, Mich. :
ProQuest,
2023

Mode of access: World Wide Web

ISBN: 9798438777410Subjects--Topical Terms:

518028
Biochemistry.
Subjects--Index Terms:

Genome editingIndex Terms--Genre/Form:

542853
Electronic books.

Engineering Virus-like Particles for the Delivery of Genome Editing Enzymes.
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500 $a Source: Dissertations Abstracts International, Volume: 84-01, Section: B.
500 $a Advisor: Turner, David L.
502 $a Thesis (Ph.D.)--University of Michigan, 2022.
504 $a Includes bibliographical references
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.
533 $a Electronic reproduction. $b Ann Arbor, Mich. : $c ProQuest, $d 2023
538 $a Mode of access: World Wide Web
650 4 $a Biochemistry. $3 518028
650 4 $a Genetics. $3 530508
650 4 $a Bioengineering. $3 657580
653 $a Genome editing
653 $a Virus-Like Particles
653 $a Enzymes
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690 $a 0369
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710 2 $a ProQuest Information and Learning Co. $3 783688
710 2 $a University of Michigan. $b Biological Chemistry. $3 3697272
773 0 $t Dissertations Abstracts International $g 84-01B.
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=29274875 $z click for full text (PQDT)