Quantification of transgene expression in GSH AAVS1 with a novel CRISPR/Cas9-based approach reveals high transcriptional variation

Inderbitzin, Anne; Loosli, Tom; Kouyos, Roger D.; Metzner, Karin J.

doi:10.1016/j.omtm.2022.06.003

Cited by 4 publications

(2 citation statements)

References 40 publications

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“…Silencing has often been observed (and less often published) for transgenes in general 65 and for transgenes similar to our KRAB-dCas9 construct inserted in the AAVS1 locus. 66 , 67 We find differences in KRAB-dCas9 expression among clones to be fairly stable across passages, although more systematic investigations would be needed to quantify this more precisely. In contrast, differences in KRAB-dCas9 expression among cells within one clone are not stable across passages, as cells sorted for high KRAB-dCas9 expression can revert back to a heterogenous population within one passage ( Figure S1 D).…”

Section: Discussionmentioning

confidence: 89%

Generation and characterization of inducible KRAB-dCas9 iPSCs from primates for cross-species CRISPRi

Edenhofer,

Térmeg,

Ohnuki

et al. 2024

iScience

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Section: Discussionmentioning

confidence: 89%

Generation and characterization of inducible KRAB-dCas9 iPSCs from primates for cross-species CRISPRi

Edenhofer,

Térmeg,

Ohnuki

et al. 2024

iScience

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“…Recently, the CRISPR/Cas9 system became popular in genetic engineering for precisely editing the chromosome at specific sites [ 21 , 22 ]. Several studies have shown that introducing the transgene into the adeno-associated virus integration site 1 (AAVS1) is safe, and the cells could maintain the transgene expression [ 23 , 24 , 25 ]. We formerly demonstrated that the mCherry gene could be delivered into the AAVS1 site of human induced pluripotent stem cells (hiPSCs).…”

Section: Introductionmentioning

confidence: 99%

Validation of Promoters and Codon Optimization on CRISPR/Cas9-Engineered Jurkat Cells Stably Expressing αRep4E3 for Interfering with HIV-1 Replication

Chupradit

Sornsuwan

Saiprayong

et al. 2022

IJMS

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Persistent and efficient therapeutic protein expression in the specific target cell is a significant concern in gene therapy. The controllable integration site, suitable promoter, and proper codon usage influence the effectiveness of the therapeutic outcome. Previously, we developed a non-immunoglobulin scaffold, alpha repeat protein (αRep4E3), as an HIV-1 RNA packaging interference system in SupT1 cells using the lentiviral gene transfer. Although the success of anti-HIV-1 activity was evidenced, the integration site is uncontrollable and may not be practical for clinical translation. In this study, we use the CRISPR/Cas9 gene editing technology to precisely knock-in αRep4E3 genes into the adeno-associated virus integration site 1 (AAVS1) safe harbor locus of the target cells. We compare the αRep4E3 expression under the regulation of three different promoters, including cytomegalovirus (CMV), human elongation factor-1 alpha (EF1α), and ubiquitin C (UbC) promoters with and without codon optimization in HEK293T cells. The results demonstrated that the EF1α promoter with codon-optimized αRep4E3mCherry showed higher protein expression than other promoters with non-optimized codons. We then performed a proof-of-concept study by knocking in the αRep4E3mCherry gene at the AAVS1 locus of the Jurkat cells. The results showed that the αRep4E3mCherry-expressing Jurkat cells exhibited anti-HIV-1 activities against HIV-1NL4-3 strain as evidenced by decreased capsid (p24) protein levels and viral genome copies as compared to the untransfected Jurkat control cells. Altogether, our study demonstrates that the αRep4E3 could interfere with the viral RNA packaging and suggests that the αRep4E3 scaffold protein could be a promising anti-viral molecule that offers a functional cure for people living with HIV-1.

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Genome Editing Tool CRISPR-Cas: Legal and Ethical Considerations for Life Science

Pandey,

Arora,

Kumar

2024

Gene Editing in Plants

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Quantification of transgene expression in GSH AAVS1 with a novel CRISPR/Cas9-based approach reveals high transcriptional variation

Cited by 4 publications

References 40 publications

Generation and characterization of inducible KRAB-dCas9 iPSCs from primates for cross-species CRISPRi

Generation and characterization of inducible KRAB-dCas9 iPSCs from primates for cross-species CRISPRi

Validation of Promoters and Codon Optimization on CRISPR/Cas9-Engineered Jurkat Cells Stably Expressing αRep4E3 for Interfering with HIV-1 Replication

Genome Editing Tool CRISPR-Cas: Legal and Ethical Considerations for Life Science

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