1. CRISPR-Cas9 targeting of MMP13 in human chondrocytes leads to significantly reduced levels of the metalloproteinase and enhanced type II collagen accumulation 
C.I. Seidl, T.A. Fulga, C.L. Murphy 
Osteoarthritis and Cartilage 
Volume 27, Issue 1, Pages (January 2019)
DOI: /j.joca
Copyright © 2018 Osteoarthritis Research Society International Terms and Conditions

2. Fig. 1
Cas9-editing strategy for human chondrocytes. ribonucleoprotein complexes (RNP), Ribonucleoprotein; N, non-edited; E, edited; ECM, extracellular matrix; KO knock-out.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2018 Osteoarthritis Research Society International Terms and Conditions

3. Fig. 2
RNP transfection of Human Articular Chondrocyte (HAC)s displays high efficiency and low toxicity. HACs at P1 in 12 well plates were left untreated (A, B) or transfected with MMP13 targeting Ribonucleoprotein complexes (RNPs, C, D) and a live-dead assay was performed 24 h post transfection. Transfection efficiency was monitored with fluorescently labelled tracrRNA within the RNP complex and uptake was visualized 6 h post transfection (E, F). To demonstrate Cas9 delivery and localization, cells transfected with RNPs containing Cas9 or without Cas9 were harvested 3 h and 20 h after transfection, separated into cytoplasmic and nuclear fractions and immunoblotted for Cas9, RNAPII and Tubulin (G). T, total cell fraction; N, nuclear fraction; C, cytoplasmic fraction. Scale bar = 100 μm.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2018 Osteoarthritis Research Society International Terms and Conditions

4. Fig. 3
Efficient editing in HACs with no off-target edits detected. A, T7E1 assay for the target area surrounding MMP13 exon2 as well as of three predicted off-targets (two intergenic and one exonic site) was performed 48 h post transfection. One OA and two healthy donors were used. B, measurement of mRNA levels of MMP13 and a protein coding predicted off-target, CNOT6 by real time qPCR. The y-axis shows relative mRNA levels normalized to RPLP0 using the delta Ct method. A paired, two-tailed Wilcoxon test was employed for statistical analysis showing the median with interquartile range. Statistical significance was set to P < 0.05. Samples from non-edited and edited cell populations were compared. Four OA and two healthy donors were used. OT, off-target; N, non-edited; E, MMP13-edited; −/+, without or with T7E1.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2018 Osteoarthritis Research Society International Terms and Conditions

5. Fig. 4
Edited cell populations retain their edits after subsequent passaging and display greatly reduced MMP13 protein levels and activity. A, T7E1 assay 3 h, 48 h and 14 days post-TF. Cells were split 2 days after TF and expanded for another 12 days. B, western blot of nuclear fractions from time points used in A. C, western blot of cell culture media of edited and non-edited cell population 22 h post IL-1β induction at the experimental endpoint of 2 weeks. Different donors were used for panels A, B and C. D, MMP13 activity assay without or with IL-1β induction comparing edited and non-edited cell populations after 2 weeks. Medians ± IQR for non-edited and edited without IL-1β are 1.438 ± 0.354 for N and 0.947 ± 0.145 for E, and after IL-1β induction  ±  for N and  ±  for E. Five OA and two healthy donors were used. E, cell counting assay using CCK-8 reagent comparing edited and non-edited cells after 2 weeks. Seven OA and two healthy donors were used. A paired, two-tailed Wilcoxon test was employed for statistical analysis and showing the median with interquartile range. Samples from non-edited and edited cell populations were compared. *, P < 0.05; N, non-edited; E, edited; −/+, without or with IL-1β at 0.1 ng/ml; relative fluorescence units (RFU), RFU; OD450, optical density at 450 nm.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2018 Osteoarthritis Research Society International Terms and Conditions

6. Fig. 5
MMP13 editing results in increased in type II collagen accumulation in spheroid HAC cultures. A, Cell culture media of non-edited and edited cells 7 days post spheroid formation was harvested and analysed by western blot. B, immunoblot for type II collagen (COL2A1) from spheroids of edited and non-edited cells harvested 7 days post spheroid formation. C, immunofluorescence for COL2A1 comparing non-edited and edited spheroids. Scale bar = 100 μm. N, non-edited; E, MMP13-edited; TRFE, transferrin.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2018 Osteoarthritis Research Society International Terms and Conditions

7. figs1
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2018 Osteoarthritis Research Society International Terms and Conditions

8. figs2
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2018 Osteoarthritis Research Society International Terms and Conditions

9. figs3
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2018 Osteoarthritis Research Society International Terms and Conditions

10. figs4
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2018 Osteoarthritis Research Society International Terms and Conditions

11. figs5
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2018 Osteoarthritis Research Society International Terms and Conditions