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CRISPR/Cas9-based Genome Editing Approaches for Gene Disruption and Application in Disease Therapy.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	CRISPR/Cas9-based Genome Editing Approaches for Gene Disruption and Application in Disease Therapy./
作者:	Zhang, Chenzi.
面頁冊數:	1 online resource (179 pages)
附註:	Source: Dissertations Abstracts International, Volume: 84-11, Section: B.
Contained By:	Dissertations Abstracts International84-11B.
標題:	Genetics. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=30545462click for full text (PQDT)
ISBN:	9798379551056

CRISPR/Cas9-based Genome Editing Approaches for Gene Disruption and Application in Disease Therapy.
Zhang, Chenzi.

CRISPR/Cas9-based Genome Editing Approaches for Gene Disruption and Application in Disease Therapy. - 1 online resource (179 pages)

Source: Dissertations Abstracts International, Volume: 84-11, Section: B.

Thesis (Ph.D.)--The Chinese University of Hong Kong (Hong Kong), 2020.

Includes bibliographical references

The recently developed CRISPR/Cas9 system can induce DNA breaks at selected sites and has made a particularly huge impact in the field of gene editing. CRISPR/Cas9 mediated homology-directed repair (HDR) enables precise genomic modification and it is widely used for gene knock-in or knockout in human cells. Whereas, CRISPR/Cas9-induced non-homologous end joining (NHEJ) is often exploited to generate loss-of-function effects in various cells and organisms. In this project, both homology-independent and -dependent knock-in approaches have been assessed using our previously developed promoter-less fluorescence reporter systems, and their potential to introduce gene disruption in human cell lines were evaluated subsequently. In particular, the potential of these CRISPR/Cas9 mediated knock-in technologies in engineering human T cells were also examined for the purpose of developing advanced Chimeric antigen receptor (CAR) T cell therapy in blood cancer treatment. We have first examined whether simultaneous knock-in of dual fluorescence reporters, through CRISPR/Cas9 induced NHEJ repair, would allow one-step multiallelic gene disruption. By knocking-in of two fluorescence reporters simultaneously at the targeted locus followed by cell enrichment via fluorescence-activated cell sorting (FACS), we successfully generated stable single-cell clones with complete disruption of ULK1 (four alleles), FAT10 (three alleles) or CtIP gene (two alleles) in hyperploidy LO2 cells. Notably, the efficiencies for complete disruption of ULK1 and FAT10 genes reached 46% and 25% in double positive LO2 cells, respectively. In the isolated single cell clones carrying dual reporters integration, we have confirmed the depletion of ULK1 and FAT10 transcripts and corresponding proteins, as well as the impaired function of these two genes. These data supported that CRISPR/Cas9-mediated homology-independent knock-in of dual reporters could be utilized for high- efficiency multiallelic gene disruption. Distinctively, when we targeted CtIP gene using the same strategy, we detected in-frame aberrant CtIP transcripts and relevant proteins from single-cell clones bearing complete disruption of CtIP gene, which suggests that robust cellular mechanisms may exist to spare essential genes from loss-of-function modifications. Furthermore, this CRISPR/Cas9 mediated gene disruption approach was applied to the recently emerged CAR-T immunotherapy to evaluate its potential in improving leukemia treatment. The insertional disruption strategy was employed to introduce chimeric antigen receptor (CAR) against B-lymphoma antigen CD19, and at the same time, disrupt T cell receptor alpha subunit constant (TRAC) gene to eliminate T-cell immunogenicity and generate universal CAR-T cells. We have compared different Cas9 delivery methods and genome editing strategies in terms of TRAC gene disruption efficiency. We found that coupling of Cas9 ribonucleoprotein (RNP) delivery with adeno-associated virus (AAV) HDR donor template would yield the best efficacy for the insertional disruption at the TRAC locus in T cell lines. We have then successfully produced CAR-T cells by blocking the TRAC gene expression while simultaneously inserting an anti-CD19 CAR fragment. Subsequent cytotoxicity assay have confirmed the cancer killing effects of these TRAC negative CAR-T cells. In a summary, through this study, we demonstrated the potentials of CRISPR/Cas9-mediated targeted knock-in technology for high efficiency gene disruption in human cells. We revealed that multiallelic gene disruption could be readily introduced through CRISPR/Cas9-induced homology-independent knock-in of dual fluorescence reporter in human cell line LO2, while the CRISPR/Cas9-based HDR reporter donor knock-in method could be applied to generate TRAC negative CAR-T cell with potential therapeutic effects.

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

Mode of access: World Wide Web

ISBN: 9798379551056Subjects--Topical Terms:

530508
Genetics.
Subjects--Index Terms:

Genome editing approachesIndex Terms--Genre/Form:

542853
Electronic books.

CRISPR/Cas9-based Genome Editing Approaches for Gene Disruption and Application in Disease Therapy.
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520 $a The recently developed CRISPR/Cas9 system can induce DNA breaks at selected sites and has made a particularly huge impact in the field of gene editing. CRISPR/Cas9 mediated homology-directed repair (HDR) enables precise genomic modification and it is widely used for gene knock-in or knockout in human cells. Whereas, CRISPR/Cas9-induced non-homologous end joining (NHEJ) is often exploited to generate loss-of-function effects in various cells and organisms. In this project, both homology-independent and -dependent knock-in approaches have been assessed using our previously developed promoter-less fluorescence reporter systems, and their potential to introduce gene disruption in human cell lines were evaluated subsequently. In particular, the potential of these CRISPR/Cas9 mediated knock-in technologies in engineering human T cells were also examined for the purpose of developing advanced Chimeric antigen receptor (CAR) T cell therapy in blood cancer treatment. We have first examined whether simultaneous knock-in of dual fluorescence reporters, through CRISPR/Cas9 induced NHEJ repair, would allow one-step multiallelic gene disruption. By knocking-in of two fluorescence reporters simultaneously at the targeted locus followed by cell enrichment via fluorescence-activated cell sorting (FACS), we successfully generated stable single-cell clones with complete disruption of ULK1 (four alleles), FAT10 (three alleles) or CtIP gene (two alleles) in hyperploidy LO2 cells. Notably, the efficiencies for complete disruption of ULK1 and FAT10 genes reached 46% and 25% in double positive LO2 cells, respectively. In the isolated single cell clones carrying dual reporters integration, we have confirmed the depletion of ULK1 and FAT10 transcripts and corresponding proteins, as well as the impaired function of these two genes. These data supported that CRISPR/Cas9-mediated homology-independent knock-in of dual reporters could be utilized for high- efficiency multiallelic gene disruption. Distinctively, when we targeted CtIP gene using the same strategy, we detected in-frame aberrant CtIP transcripts and relevant proteins from single-cell clones bearing complete disruption of CtIP gene, which suggests that robust cellular mechanisms may exist to spare essential genes from loss-of-function modifications. Furthermore, this CRISPR/Cas9 mediated gene disruption approach was applied to the recently emerged CAR-T immunotherapy to evaluate its potential in improving leukemia treatment. The insertional disruption strategy was employed to introduce chimeric antigen receptor (CAR) against B-lymphoma antigen CD19, and at the same time, disrupt T cell receptor alpha subunit constant (TRAC) gene to eliminate T-cell immunogenicity and generate universal CAR-T cells. We have compared different Cas9 delivery methods and genome editing strategies in terms of TRAC gene disruption efficiency. We found that coupling of Cas9 ribonucleoprotein (RNP) delivery with adeno-associated virus (AAV) HDR donor template would yield the best efficacy for the insertional disruption at the TRAC locus in T cell lines. We have then successfully produced CAR-T cells by blocking the TRAC gene expression while simultaneously inserting an anti-CD19 CAR fragment. Subsequent cytotoxicity assay have confirmed the cancer killing effects of these TRAC negative CAR-T cells. In a summary, through this study, we demonstrated the potentials of CRISPR/Cas9-mediated targeted knock-in technology for high efficiency gene disruption in human cells. We revealed that multiallelic gene disruption could be readily introduced through CRISPR/Cas9-induced homology-independent knock-in of dual fluorescence reporter in human cell line LO2, while the CRISPR/Cas9-based HDR reporter donor knock-in method could be applied to generate TRAC negative CAR-T cell with potential therapeutic effects.
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