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. 2024 Mar 11;26(1):66.
doi: 10.1186/s13075-024-03294-w.

Streamlined, single-step non-viral CRISPR-Cas9 knockout strategy enhances gene editing efficiency in primary human chondrocyte populations

Simone Ponta #  1 , Angela Bonato #  1 , Philipp Neidenbach  2 , Valentino F Bruhin  2 , Alexis Laurent  3 , Lee Ann Applegate  3 , Marcy Zenobi-Wong  1 , Goncalo Barreto  4   5   6
Affiliations

Streamlined, single-step non-viral CRISPR-Cas9 knockout strategy enhances gene editing efficiency in primary human chondrocyte populations

Simone Ponta et al. Arthritis Res Ther. .

Abstract

Background: CRISPR-Cas9-based genome engineering represents a powerful therapeutic tool for cartilage tissue engineering and for understanding molecular pathways driving cartilage diseases. However, primary chondrocytes are difficult to transfect and rapidly dedifferentiate during monolayer (2D) cell culture, making the lengthy expansion of a single-cell-derived edited clonal population not feasible. For this reason, functional genetics studies focused on cartilage and rheumatic diseases have long been carried out in cellular models that poorly recapitulate the native molecular properties of human cartilaginous tissue (e.g., cell lines, induced pluripotent stem cells). Here, we set out to develop a non-viral CRISPR-Cas9, bulk-gene editing method suitable for chondrocyte populations from different cartilaginous sources.

Methods: We screened electroporation and lipid nanoparticles for ribonucleoprotein (RNP) delivery in primary polydactyly chondrocytes, and optimized RNP reagents assembly. We knocked out RELA (also known as p65), a subunit of the nuclear factor kappa B (NF-κB), in polydactyly chondrocytes and further characterized knockout (KO) cells with RT-qPCR and Western Blot. We tested RELA KO in chondrocytes from diverse cartilaginous sources and characterized their phenotype with RT-qPCR. We examined the chondrogenic potential of wild-type (WT) and KO cell pellets in presence and absence of interleukin-1β (IL-1β).

Results: We established electroporation as the optimal transfection technique for chondrocytes enhancing transfection and editing efficiency, while preserving high cell viability. We knocked out RELA with an unprecedented efficiency of ~90%, confirming lower inflammatory pathways activation upon IL-1β stimulation compared to unedited cells. Our protocol could be easily transferred to primary human chondrocytes harvested from osteoarthritis (OA) patients, human FE002 chondroprogenitor cells, bovine chondrocytes, and a human chondrocyte cell line, achieving comparable mean RELA KO editing levels using the same protocol. All KO pellets from primary human chondrocytes retained chondrogenic ability equivalent to WT cells, and additionally displayed enhanced matrix retention under inflamed conditions.

Conclusions: We showcased the applicability of our bulk gene editing method to develop effective autologous and allogeneic off-the-shelf gene therapies strategies and to enable functional genetics studies in human chondrocytes to unravel molecular mechanisms of cartilage diseases.

Keywords: CRISPR-Cas9; Gene editing; NF-κB; Primary chondrocytes; RELA.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Overview of the experimental workflow outlined in this study. A Transfection optimization in primary polydactyly chondrocytes was performed by delivering a green fluorescent protein (GFP)-labelled Cas9-RNP targeting the housekeeping hypoxanthine guanine phosphoribosyltransferase (HPRT) gene, comparing electroporation and lipid nanoparticle delivery. After 24 h, microscopy imaging was used to infer transfection-associated cytotoxicity and efficiency, by counting live, dead and GFP+ cells, respectively. At 48 h post-delivery, DNA was extracted and the HPRT locus was amplified by polymerase chain reaction (PCR). Upon denaturation and re-annealing, mismatched heteroduplexes arising from base-pairing of WT and edited alleles were cut using the T7 endonuclease 1 (T7E1) enzyme. The generated fragments were run on a 1.2% agarose gel and editing efficiency was calculated. B Reagents optimization was carried out by testing three different Cas9 enzymes and two gRNA formulations, respectively. Editing efficiency at the HPRT locus in polydactyly chondrocytes was validated with Sanger sequencing. C Using our optimized parameters and reagents, we generated bulk-edited chondrocyte populations harboring a RELA KO. RELA is an essential component of the NF-κB complex, which regulates the activation of several pro-inflammatory cellular pathways (e.g., interleukin-1 beta (IL-1β), MMP13 and tumor necrosis factor alpha (TNFα)). Besides calculating editing efficiency, we further characterized KO polydactyly chondrocytes with quantitative reverse transcription PCR (RT-qPCR) and Western Blot. Applicability and reproducibility of our KO gene editing technique was further tested in OA-patient-derived chondrocytes, FE002 primary chondroprogenitors, a human chondrocyte cell line and bovine chondrocytes, with RELA KO efficiency calculated using Sanger sequencing and phenotype validated via qPCR. D WT and KO pellets for all cell types were cultured for 3 weeks in chondrogenic media, with and without the addition of 10 ng/ml of IL-1β throughout the last week of culture. Extracellular matrix (ECM) deposition was assessed using histological staining for detecting glycosaminoglycans (GAGs) and immunostaining to verify the deposition of collagen type I and type II fibers
Fig. 2
Fig. 2
Electroporation leads to more effective transfection and HPRT editing efficiency compared to lipid nanoparticle delivery. Cell viability, transfection and editing efficiencies were calculated upon Cas9-RNP delivery with A the lipid nanoparticles Lipofectamine 3000™, Lipofectamine™ RNAiMAX and FuGENE® or the B Neon™ electroporation system, by testing different ranges of voltage, milliseconds or applied pulses, respectively. C Neon™ electroporation program optimization, calculating % of live cells, % of GFP+ cells and T7E1 efficiency. Non-transfected cells were used as viability controls, while cells transfected with an incomplete RNP, consisting of a Cas9-GFP without gRNA, were used as transfection and editing efficiency controls. Data are represented as mean ± standard deviation of 3 technical replicates from one donor (n = 3). Statistical significance was determined using one-way ANOVA with a Tukey’s multiple comparisons correction (* p < 0.05, ** p < 0.01, and *** p < 0.001)
Fig. 3
Fig. 3
TrueCut™ Cas9 v2 and sgRNA provided by Synthego lead to high HPRT editing efficiency. Editing efficiency of the HPRT locus was calculated upon Cas9-RNP delivery testing while comparing A three different Cas9 manufacturers, B the gRNA synthesized as either a cr:tracrRNA complex or sgRNA at various Cas9:gRNA ratios and C two distinct sgRNA providers, testing multiple Cas9:sgRNA ratios. Data are represented as mean ± standard deviation of 3 technical replicates from one donor (n = 3). Statistical significance was determined using one-way ANOVA with a Tukey’s multiple comparisons correction (** p < 0.01, and **** p < 0.0001)
Fig. 4
Fig. 4
Sanger sequencing reveals highly efficient RELA KO in primary human polydactyly chondrocytes, and expression of NF-κB-dependent inflammatory pathways is lower in KO cells. A Editing efficiency of five different sgRNAs targeting the RELA gene at different locations. B Off-target effects of the best-performing sgRNAs #1, #2 and #5 at the top five in silico-predicted most probable off-target sites. Data are represented as mean ± standard deviation of 3 biological replicates from three donors (n = 3). Statistical significance was determined using a nonparametric one-way ANOVA (* p < 0.05, ** p < 0.01, and **** p < 0.0001). C RT-qPCR of selected inflammatory pathways acting downstream of NF-κB in WT and KO cells following stimulation with IL-1β for 16 h. D Western blot and quantification of RELA in WT and KO cells treated with IL-1β for 30 min compared to housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels. RT-qPCR data are represented as mean ± standard deviation of 3 technical replicates from three biological donors (n = 9). Western Blot data are represented as mean ± standard deviation of 3 biological replicates from three donors (n = 3). Statistical significance was determined using a nonparametric Mann–Whitney t-test
Fig. 5
Fig. 5
RELA KO is reproducible in a wide variety of primary chondrocyte populations and in a chondrocyte cell line with comparably high editing efficiency. (A) Editing efficiency and (B) cellular viability data of OA chondrocytes, human FE002 primary chondroprogenitors, the C28/I2 cell line and bovine chondrocytes edited with sgRNA #1 to induce a RELA KO. Cells mock-transfected with an incomplete RNP (i.e., Cas9 only, no sgRNA) were used as a control. RT-qPCR of selected inflammatory pathways acting downstream of NF-κB in (C) human and (D) bovine WT and KO cells following stimulation with IL-1β for 16 h. Data are represented as mean ± standard deviation of 3 technical replicates for bovine chondrocytes (n = 3) and the C28/I2 cell line (n = 3), of 3 technical replicates from two primary chondroprogenitor donors (n = 6), and of 3 technical replicates from three OA chondrocyte donors (n = 9). Statistical significance was determined using an unpaired t-test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001)
Fig. 6
Fig. 6
RELA KO chondrocyte pellets display ECM deposition compared to WT cells, and they are more resistant to IL-1β induced catabolism. Infant articular cartilage was used as a A positive and B negative histological control. Safranin O, collagen type I and II immunostaining of C polydactyly chondrocytes, D FE002 chondroprogenitor cells, and E OA chondrocyte pellets in an untreated condition after 21 days of culture in chondrogenic media. Safranin O, collagen type I and II immunostaining of F polydactyly chondrocytes, G FE002 chondroprogenitor cells, and H OA chondrocytes treated with 10 ng/ml of IL-1β in the last week of culture in chondrogenic media. I-K Quantification of the relative staining intensity. 20X pictures scale bar, 100 µm; low magnification 2X scale bar, 500 µm. One donor per cell type is showed as a representative example
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