Aim. Comparison of native and chemically modified synthetic crRNAs in complex with MAD7 nuclease for reporter knockout in eukaryotic cell lines. Evaluation of alternative crRNA array formats on MAD7-mediated multiplexed gene editing in eukaryotic cells. Conclusions. We demonstrated that the MAD7-based editing activity depends on synthetic crRNA modifications, favoring stabilizing modifications at both 5`- and 3`-ends of crRNA. Efficient crRNA array processing in single-cistron format requires 5`-terminal sequences from full-DR configuration. Further optimization of all components is required to increase system activity in mammalian cells.