Jetcrispr

ENH5 deletion lines were generated in teloHAECs (ATCC) using a modified IDT ALT-R Ribonucleoprotein (RNP) Protocol. Four CRISPR RNAs (crRNA) were designed to flank the region containing ENH5 using IDT’s Custom ALT-R CRISPR-Cas9 guide RNA design tool (table S15). Guides were selected on the basis of optimizing location and predicted efficacy while minimizing the possibility of off-target effects. The four crRNAs were annealed separately to tracrRNA to create four complete guide RNAs. RNPs were complexed by incubating the guides with purified Alt-R S.p. Cas9 Nuclease V3 (IDT, 1081058). Guides and RNPs were generated as described in the IDT ALT-R RNP Protocol. The pool of four RNPs was transfected into teloHAECs using jetCRISPR (PolyPlus, 502-07). Because of the difficulty of transfecting endothelial cells, this transfection was serially performed two to four times as described in (82 (link)) as cells were expanded and periodically checked by PCR for the presence of a deletion band (table S4). Once a strong deletion band was evident, the pool of edited cells was single-cell sorted into a 96-well plate on the FACSARIAIII at the University of Chicago Flow Core. Clones were genotyped using primers designed to flank the deletion site (table S16). Of all clones isolated, three KO clones and three WT controls from the same pool of cells were randomly selected for further analysis.

Roswell Park Memorial Institute (RPMI)-1640 medium, Dulbecco’s modified Eagle’s medium containing growth factor F-12 (DMEM/F-12), Iscove’s modified Dulbecco’s medium (IMDM), fetal bovine serum (FBS), Opti-MEM (minimal essential medium), L-glutamine, penicillin/streptomycin (P/S) solution (5,000 IU/mL penicillin and 5,000 μg/mL streptomycin), Dulbecco’s phosphate-buffered saline without Ca²⁺/Mg²⁺ (DPBS−), geneticin (G418) antibiotic, TO-PRO3 iodide (1 mM), 10 kDa Alexa Fluor 647 dextran, FD250, and RNAiMAX were all obtained Life Technologies (Merelbeke, Belgium). JetCRISPR was received from Polyplus (Illkirch, France). Alt-R S.p. Cas9 Nuclease V3, Alt-R custom crRNAs, Alt-R CRISPR Negative Control crRNA #1, and Alt-R tracrRNA were purchased from Integrated DNA Technologies (Leuven, Belgium). sgRNAs were purchased from either Integrated DNA Technologies (PD-1) or Synthego (WAS). Table S1 lists the different crRNA, sgRNA, and primer sequences used throughout the manuscript.

The inhibition of MMP9 gene was carried out by transfecting the gRNAs and Cas9 protein (IDT, Singapore) into A431 cells. Ribonucleoprotein (RNP) complex was formed by mixing equimolar (ratio 1 : 1) of a gRNA and Cas9 protein. Briefly, in each well, 16.5 µl of 1 µm gRNA and 16.5 µl of 1 µm Cas9 protein were added to 17 µl of Opti-MEM medium (Gibco, USA). The mixture was then resuspended and incubated at room temperature for 10 min. After that, 1.2 µl of jet CRISPR (Polyplus-transfection, France) was added to the mixture, resuspended, and incubated at room temperature for 15 min. The transfection solution was prepared in a reservoir before being distributed into each well. Then, 1 × 10⁵ A431 cells in 500 µl medium was seeded to each well. The plate was then incubated at 37°C for 48 hours.