Optimization of Vectors and Targeting Strategies Including GoldenBraid and Genome Editing Tools: GoldenBraid Assembly of Multiplex CRISPR /Cas12a Guide RNAs for Gene Editing in Nicotiana benthamiana.

New breeding techniques, especially CRISPR/Cas, could facilitate the expansion and diversification of molecular farming crops by speeding up the introduction of new traits that improve their value as biofactories. One of the main advantages of CRISPR/Cas is its ability to target multiple loci simultaneously, a key feature known as multiplexing. This characteristic is especially relevant for polyploid species, as it is the case of Nicotiana benthamiana and other species of the same genus widely used in molecular farming. Here, we describe in detail the making of a multiplex DNA construct for genome editing in N. benthamiana using the GoldenBraid modular cloning platform. In this case, the procedure is adapted for the requirements of LbCas12a (Lachnospiraceae bacterium Cas12a), a nuclease whose cloning strategy differs from that of the more often used SpCas9 (Streptococcus pyogenes Cas9) enzyme. LbCas12a-mediated edition has several advantages, as its high editing efficiency, described for different plant species, and its T/A-rich PAM sequence, which expands the range of genomic loci that can be targeted by site-specific nucleases. The protocol also includes recommendations for the selection of protospacer sequences and indications for the analysis of editing results.