Design and Derivation of Multi‐Reporter Pluripotent Stem Cell Lines via CRISPR/Cas9n‐Mediated Homology‐Directed Repair

Dettmer, Rabea; Naujok, Ortwin

doi:10.1002/cpsc.116

Cited by 3 publications

(3 citation statements)

References 32 publications

(27 reference statements)

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“…New breakthroughs in CRISPR/Cas9 gene editing have allowed researchers to manipulate this system to induce the desired mutation. For example, using an HDR template allows for the incorporation of an in-frame stop codon, permitting specific knockout of the target gene in all cells 53 – 55 . Our designed dual-sgRNA CRISPR-Cas9 plasmid generates two cuts (adjacent to each other) on the genomic DNA, preventing off-target effects and improving genomic editing specificity and therapeutic accuracy.…”

Section: Discussionmentioning

confidence: 99%

Dual-sgRNA CRISPR/Cas9 knockout of PD-L1 in human U87 glioblastoma tumor cells inhibits proliferation, invasion, and tumor-associated macrophage polarization

Fierro

DiPasquale

Perez

et al. 2022

Sci Rep

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Programmed death ligand 1 (PD-L1) plays a key role in glioblastoma multiforme (GBM) immunosuppression, vitality, proliferation, and migration, and is therefore a promising target for treating GBM. CRISPR/Cas9-mediated genomic editing can delete both cell surface and intracellular PD-L1. This systemic deliverable genomic PD-L1 deletion system can be used as an effective anti-GBM therapy by inhibiting tumor growth and migration, and overcoming immunosuppression. To target PD-L1 for CRISPR/Cas9 gene editing, we first identified two single guide RNA (sgRNA) sequences located on PD-L1 exon 3. The first sgRNA recognizes the forward strand of human PD-L1 near the beginning of exon 3 that allows editing by Cas9 at approximately base pair 82 (g82). The second sgRNA recognizes the forward strand of exon 3 that directs cutting at base pair 165 (g165). A homology-directed repair template (HDR) combined with the dual-sgRNAs was used to improve PD-L1 knockout specificity and efficiency. sgRNAs g82 and g165 were cloned into the multiplex CRISPR/Cas9 assembly system and co-transfected with the HDR template in human U87 GBM cells (g82/165 + HDR). T7E1 analysis suggests that the dual-sgRNA CRISPR/Cas9 strategy with a repair template was capable of editing the genomic level of PD-L1. This was further confirmed by examining PD-L1 protein levels by western blot and immunofluorescence assays. Western blot analysis showed that the dual-sgRNAs with the repair template caused a 64% reduction of PD-L1 protein levels in U87 cells, while immunostaining showed a significant reduction of intracellular PD-L1. PD-L1 deletion inhibited proliferation, growth, invasion and migration of U87 cells, indicating intracellular PD-L1 is necessary for tumor progression. Importantly, U87 cells treated with g82/165 + HDR polarized tumor-associated macrophages (TAM) toward an M1 phenotype, as indicated by an increase in TNF-α and a decrease in IL-4 secretions. This was further confirmed with flow cytometry that showed an increase in the M1 markers Ly6C + and CD80 +, and a decrease in the M2 marker CD206 + both in vitro and in vivo. Utilizing dual-sgRNAs and an HDR template with the CRISPR/Cas9 gene-editing system is a promising avenue for the treatment of GBM.

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Section: Discussionmentioning

confidence: 99%

Dual-sgRNA CRISPR/Cas9 knockout of PD-L1 in human U87 glioblastoma tumor cells inhibits proliferation, invasion, and tumor-associated macrophage polarization

Fierro

DiPasquale

Perez

et al. 2022

Sci Rep

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“…SgRNA sequence details are available in Supplementary table 1 . Reporter genes were introduced by HDR [ 17 ]. A scheme of the repair vectors is depicted in Figs.…”

Section: Methodsmentioning

confidence: 99%

New hPSC SOX9 and INS Reporter Cell Lines Facilitate the Observation and Optimization of Differentiation into Insulin-Producing Cells

Dettmer

Niwolik

Mehmeti

et al. 2021

Stem Cell Rev and Rep

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Differentiation of human pluripotent stem cells into insulin-producing stem cell-derived beta cells harbors great potential for research and therapy of diabetes. SOX9 plays a crucial role during development of the pancreas and particularly in the development of insulin-producing cells as SOX9+ cells form the source for NEUROG3+ endocrine progenitor cells. For the purpose of easy monitoring of differentiation efficiencies into pancreatic progenitors and insulin-producing cells, we generated new reporter lines by knocking in a P2A-H-2Kk-F2A-GFP2 reporter gene into the SOX9-locus and a P2A-mCherry reporter gene into the INS-locus mediated by CRISPR/CAS9-technology. The knock-ins enabled co-expression of the endogenous and reporter genes and report on the endogenous gene expression. Furthermore, FACS and MACS enabled the purification of pancreatic progenitors and insulin-producing cells. Using these cell lines, we established a new differentiation protocol geared towards SOX9+ cells to efficiently drive human pluripotent stem cells into glucose-responsive beta cells. Our new protocol offers an alternative route towards stem cell-derived beta cells, pointing out the importance of Wnt/beta-catenin inhibition and the efficacy of EGF for the development of pancreatic progenitors, as well as the significance of 3D culture for the functionality of the generated beta cells. Graphic Abstract

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“…Putative sgRNAs were calculated with CCTop (https://cctop.cos.uni-heidelberg.de/) [45].The sgRNA for the SOX9 locus was cloned into the pLKO5.U6 vector [46] and the sgRNAs, two nickase pairs, for the INS locus were cloned into the pX335-U6-Chimeric_BB-CBh-hSpCas9n vector [47] (Supplementary table 1). Reporter genes were introduced by HDR [48]. A scheme of the repair vectors is presented in Figure 1A/4A.…”

Section: Generation Of Hpsc Reporter Cell Linesmentioning

confidence: 99%

Pluripotent stem cell SOX9 and INS reporters facilitate differentiation into insulin-producing cells

Dettmer

Niwolik

Mehmeti

et al. 2021

Preprint

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Differentiation of human pluripotent stem cells into insulin-producing stem cell-derived beta cells harbors great potential for research and therapy of diabetes. The SOX9 gene plays a crucial role during development of the pancreas and particularly in the development of insulin-producing cells as SOX9+ cells form the source for NEUROG3+ endocrine progenitor cells. For the purpose of easy monitoring of differentiation efficiencies into pancreatic progenitors and insulin-producing cells, we generated new reporter lines by knocking in a P2A-H-2Kk-F2A-GFP2 reporter genes into the SOX9 locus and a P2A-mCherry reporter gene into the INS locus mediated by CRISPR/CAS9-technology. The knock-ins enable co-expression of the endogenous genes and reporter genes, report the endogenous gene expression and enable the purification of pancreatic progenitors and insulin-producing cells using FACS or MACS. Using these cell lines we established a new differentiation protocol geared towards SOX9+ cells to efficiently drive human pluripotent stem cells into glucose-responsive beta cells.

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Design and Derivation of Multi‐Reporter Pluripotent Stem Cell Lines via CRISPR/Cas9n‐Mediated Homology‐Directed Repair

Cited by 3 publications

References 32 publications

Dual-sgRNA CRISPR/Cas9 knockout of PD-L1 in human U87 glioblastoma tumor cells inhibits proliferation, invasion, and tumor-associated macrophage polarization

Dual-sgRNA CRISPR/Cas9 knockout of PD-L1 in human U87 glioblastoma tumor cells inhibits proliferation, invasion, and tumor-associated macrophage polarization

New hPSC SOX9 and INS Reporter Cell Lines Facilitate the Observation and Optimization of Differentiation into Insulin-Producing Cells

Pluripotent stem cell SOX9 and INS reporters facilitate differentiation into insulin-producing cells

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