Generation of multiplex tRNA-gRNA constructs for Marchantia polymorpha CRISPR v1

This protocol describes a Marchantia polymorpha specific modification of the Xie et al 2015 protocol(https://doi.org/10.1073/pnas.1420294112) for the synthesis of multicomplex tRNA-gRNA modules by Golden Gate Assembly/Loop Assembly. It has 3 main steps: A. Primer design and PCR amplification of tRNA-gRNA fragments using the pGTR plasmid as the template. B. Loop assembly cloning of the amplified tRNA-gRNA fragments into an L1 vector,to combine them with the MpU6 promoter C. Loop assembly cloning into the L2 pCsA acceptor vector to combine the MpU6::tRNA-gRNA unit with the transcription unit for Cas9 expression