Fig. 2.

Construction of the gRNA expression plasmids pUDP003 and pUDP004. In vitro “Gibson” assembly [39] of functional parts containing an amdS selection marker cassette, a synthetic pangenomic yeast replication origin panARSopt, an E. coli replication origin from pBR322 and the bla gene conferring resistance to β-lactam antibiotics and a gRNA expression cassette using 60 bp synthetic homologous recombination sequences into pUDP003 and with the addition of a fragment carrying a Spcas9 expression part into pUDP004. The ribozymes flanked gRNA is next directionally inserted into pUDP003 or pUDP004 using BsaI digestion and ligation yielding the gRNA expressing plasmids pUDP010 and pUDP012 respectively