This protocol explains how to design clone the guide RNA (gRNA) sequence into the L2 plasmids ready to accept the gRNA sequence by cloning with SapI. We have two versions of the plasmid, with or without Cas9: the L2_gRNA-Cas9-CsA and L2_gRNA-CsA plasmids. With L2_gRNA-Cas9-CsA you transform wild-type sporelings.With L2_gRNA-CsA you transform sporelings of a line expressing Cas9.