Golden Gate cloning method enables assembly of CRISPR modules in various combinations.

Cas9 alleles, promoters and terminators were cloned into the indicated Level 0 acceptor vectors as described in Materials and Methods and were assembled in Level 1 acceptor vector pICH47742. sgRNAs targeting AtADH1 were amplified by PCR and assembled with the U6-26 promoter vector pICSL90002 in the same manner. Both Cas9 and sgRNA expression units were assembled in Level 2 acceptors pAGM4723 (not containing an overdrive sequence) or pICSL4723 (containing an overdrive) along with a Glufosinate resistance plant selectable marker. An end-linker pICH41766 (EL2;3) was used to link the sgRNA expression unit to the Level 2 acceptor vector. For a “head-to-head orientation” of the sgRNA and Cas9 expression cassettes, Cas9 allele, promoter and terminator were assembled into pICH47811 instead of pICH47742.