Engineering of crRNA sequences for MAD7 cleavage efficiency optimization

Abstract

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.