Active fusions of Cas9 orthologs

Josipović, Goran; Zoldoš, Vlatka; Vojta, Aleksandar

doi:10.1016/j.jbiotec.2019.05.306

Cited by 12 publications

(2 citation statements)

References 32 publications

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“…90 As a last step, the Cas9 RNP has to enter the nucleus to reach its target site and for this reasons nuclear localization signal (NLS)-fused Cas9 is commonly used. 91 As discussed above, the duration of intracellular persistence depends on the stability of the genetic 'blue prints' and degradation of encoded protein. For genome editing applications, the CRISPR/Cas components are only transiently required until the intended genome modification has occurred; an additional presence in the cell generally is not favored, since the risk of off-target events can increase with the duration of presence in the cell.…”

Section: Comparison Of Different Formats Of Crispr/cas9mentioning

confidence: 97%

Non-viral delivery of the CRISPR/Cas system: DNAversusRNAversusRNP

2022

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Section: Comparison Of Different Formats Of Crispr/cas9mentioning

confidence: 97%

Non-viral delivery of the CRISPR/Cas system: DNAversusRNAversusRNP

2022

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“…Expression plasmids encoding gene-specific guide RNA (gRNA) molecules were constructed in the multi-guide system described by Josipovićet al (21). Three gRNA molecules were cloned individually in a backbone plasmid pSgMx-A or pSgMx-G (where x represents the order of gRNA molecules; 1,2,3 and A or G represents Cas9 ortholog (dSaCas9 or dSpCas9 respectively) it recognizes) (22) for each gene: RUNX1, RUNX3, SPINK4 and ELL2. gRNA molecules for RUNX1, RUNX3, ELL2 and SPINK4 were then cloned in pSgx3 as modular "multiguide" molecules.…”

Section: Plasmid Constructsmentioning

confidence: 99%

Effects of Estradiol on Immunoglobulin G Glycosylation: Mapping of the Downstream Signaling Mechanism

et al. 2021

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Glycans attached to immunoglobulin G (IgG) directly affect this antibody effector functions and regulate inflammation at several levels. The composition of IgG glycome changes significantly with age. In women, the most notable change coincides with the perimenopausal period. Aiming to investigate the effect of estrogen on IgG glycosylation, we analysed IgG and total serum glycomes in 36 healthy premenopausal women enrolled in a randomized controlled trial of the gonadotropin-releasing hormone analogue (GnRHAG) leuprolide acetate to lower gonadal steroids to postmenopausal levels and then randomized to transdermal placebo or estradiol (E2) patch. The suppression of gonadal hormones induced significant changes in the IgG glycome, while E2 supplementation was sufficient to prevent changes. The observed glycan changes suggest that depletion of E2 primarily affects B cell glycosylation, while liver glycosylation stays mostly unchanged. To determine whether previously identified IgG GWAS hits RUNX1, RUNX3, SPINK4, and ELL2 are involved in downstream signaling mechanisms, linking E2 with IgG glycosylation, we used the FreeStyle 293-F transient system expressing IgG antibodies with stably integrated CRISPR/dCas9 expression cassettes for gene up- and downregulation. RUNX3 and SPINK4 upregulation using dCas9-VPR resulted in a decreased IgG galactosylation and, in the case of RUNX3, a concomitant increase in IgG agalactosylation.

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