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.