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Fluorescent labeling of CRISPR/Cas9 RNP for gene knockout in HSPCs and iPSCs reveals an essential role for GADD45b in stress response

Masoud Nasri, Perihan Mir, Benjamin Dannenmann, Diana Amend, Tessa Skroblyn, Yun Xu, Klaus Schulze-Osthoff, Maksim Klimiankou, Karl Welte and Julia Skokowa

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Article Figures & Data

Figures

  • Figure 1.

    Scheme of CRISPR/Cas9–gRNA RNP labeling and cell transfection. (A) crRNA and tracrRNA were annealed at room temperature for 10 minutes. The resulting gRNA was labeled with fluorescein- or CX-rhodamine–coupled Label IT Tracker labeling reagent. The fluorescent GADD45B-targeting gRNA was assembled with recombinant Cas9 protein prior to transfection to assemble an active CRISPR/Cas9–gRNA RNP complex targeting human GADD45B. Cells were transfected with TransIT-X2 Transfection Reagent or by using the Amaxa Nucleofector System and were incubated for 24 hours before sorting the CX-rhodamine+ or fluorescein+ cells using a BD FACSAria II. After sorting, some of the cells were used for a single-cell culture, and the rest were used for DNA isolation or cell-based assays. (B) Virtual gel of an Agilent Bioanalyzer analysis revealing no difference in the size or quality of labeled gRNA compared with unlabeled gRNA. (C) GADD45B was targeted using gRNA (highlighted in red), which inserts a double-strand break at NM_015675.3 exon 1, 31 bp after ATG; NP_056490.2, p.N11.

  • Figure 2.

    Transfection- and genome-editing efficiency in different cell types using CX-rhodamine–labeled CRISPR/Cas9–gRNA RNP targeting GADD45B. (A) HEK293FT cells were transfected with CX-rhodamine–labeled GADD45B-targeting CRISPR/Cas9–gRNA RNP. Fluorescence signal could be detected for up to 72 hours posttransfection. Representative images of 3 experiments are shown. (B) HEK293FT cells, Jurkat cells, human iPSCs, and CD34+ cells were transfected with labeled GADD45B-targeting CRISPR/Cas9–gRNA RNP. At 24 hours posttransfection, cells were harvested and measured for transfection efficiency using a BD FACSCanto II flow cytometer. Representative line graphs of 3 independent experiments are shown. (C) HEK293FT cells, Jurkat cells, human iPSCs, and CD34+ HSPCs were transfected with unlabeled or labeled GADD45B-targeting CRISPR/Cas9–gRNA RNP and analyzed for gene-modification efficiency using a TIDE assay. (D) Jurkat cells, human iPSCs, and CD34+ HSPCs were transfected with CX-rhodamine–labeled GADD45B-targeting CRISPR/Cas9–gRNA RNP and sorted 24 hours posttransfection using a flow cytometer. Genomic DNA was isolated 48 hours posttransfection from the total population of transfected cells and from sorted CX-rhodamine+ or fluorescein+ cells. TIDE assay analysis showed significantly higher gene modification efficiency in CX-rhodamine+ cells. Data in panels C and D are mean ± standard deviations derived from 3 (HEK293FT cells, Jurkat cells, CD34+ HSPCs) or 4 (iPSCs) independent experiments. *P ≤ .05, **P ≤ .01, Student t test. ns, not significant.

  • Figure 3.

    GADD45B knockout leads to reduced cell viability and increased UV-induced cellular stress. (A) Cell viability of HEK293FT cells, Jurkat cells, iPSCs, and CD34+ HSPCs, transfected with labeled tracr-Cas9 RNP (nontarget RNP) or with labeled GADD45B-targeting CRISPR/Cas9–gRNA RNP, was measured after exposing the cells to UV for 5 minutes, followed by 2 hours of additional incubation. Relative viability of nonirradiated control cells was set as 1.0. (B) CD34+ HSPCs were transfected with fluorescein-labeled GADD45B-targeting CRISPR/Cas9–gRNA RNP. After 48 hours, the cells were exposed to UV irradiation for 5 minutes. Following 2 hours of further incubation, intracellular γH2AX (phospho-Ser139) levels were measured by flow cytometry. (C) Jurkat cells were transfected with CX-rhodamine–labeled GADD45B-targeting CRISPR/Cas9–gRNA RNP. After 48 hours, the total population was exposed to UV irradiation for 5 minutes, followed by 2 hours of incubation before performing intracellular staining and FACS analysis for the DNA damage marker γH2AX (phospho-Ser139). (D) mtDNA damage (left panel) and nuclear DNA damage in the GAPDH locus (middle panel) and TP53 locus (right panel) were quantified in Jurkat control cells and a GADD45B−/− Jurkat clone using the LORD-Q method. Data are mean ± standard deviation from 3 (A-B) or 2 (C-D) independent experiments, each performed in duplicates. *P ≤ .05, **P ≤ .01, Student t test.

  • Figure 4.

    Heterozygous and homozygous GADD45B knockout in human iPSCs results in high levels of DNA damage. (A) Pluripotency state of GADD45B+/− and GADD45B−/− iPSCs was assessed by real-time quantitative PCR and compared with validated healthy donor–derived human iPSCs expressing wild-type GADD45B. (B) GADD45B wild-type, GADD45B+/−, and GADD45B−/− iPSCs were irradiated with UV light for 5 minutes, incubated under cell culture conditions for 2 hours, and stained for intracellular γH2AX (phospho-Ser139). DNA damage in GADD45B wild-type and GADD45B heterozygous-knockout (C) or homozygous-knockout (D) iPSCs was quantified by the LORD-Q method. Cells were analyzed for mtDNA damage and nuclear DNA damage in the GAPDH and TP53 gene loci. All data are mean ± standard deviation derived from 3 independent experiments. *P ≤ .05, **P ≤ .01, ***P ≤ .001, Student t test.