Fig. 4.

Toolkit cloning strategy. Toolkit main cloning routes: A = level 1 oligo cloning to give guide cassettes (black boxes with red outline), B = Up to 4 level 1 guides directly cloned into level 2 accepter, C = more than 4 guides cloned into level 2 accepter via intermediate level M step. Thin arrows represent complementary oligonucleotides that will later transcribe as the protospacer. Thick arrows show the direction of cloning. U6/3 = U6 or U3 promoter (Note – the invariable DR of Cas12a guide and sgRNA of Cas9 guide are not shown in level 1 guide accepters, but they are present outside of the BsaI-BsaI fragment. LacZ = blue/white colour selection marker for cloning, RFP = red fluorescent protein for colour selection during cloning, BsaI = restriction site, BpiI = restriction site. Triangles indicate GoldenGate cloning site cut points, HptII = hygromycin phosphotransferase plant selection marker, Cas = Cas9 or Cas12a nuclease cassette