Two-dimensional materials have risen in popularity as a desired material for membrane in the last decade. Nanoporous single-layer graphene, prepared by incorporating subnanometer vacancy defects in the graphene lattice, is highly promising for high flux gas separation because the resistance to diffuse is controlled by a single transition state at the nanopore [1–3]. Molecular sieving resolution (MSR), defined as the difference in the kinetic diameters of molecules to be separated, of a fraction of an angstrom has been predicted, allowing separation of industrially-relevant mixtures such as CO2/N2, CO2/CH4, O2/N2, etc, allowing graphene-based membranes to compete with those from zeolites and carbon molecular sieves. However, the realization of single-layer graphene membranes for gas separation has been hampered because of the difficulty in controlling the nucleation and growth of vacancy defects in graphene.