2.3. U6 promoter/sgRNA entry vector constructs

To generate pENTR‐PpU6‐sgRNA‐L1L2, the first Gateway entry vector for the P. patens vector system, we amplified the PpU6 and sgRNA fragments with two separate PCRs. For the PpU6 fragment, we used a forward primer containing an AscI site and a reverse primer containing two inverted BsaI sites at the 5′ ends (Table S1). Similarly, for the sgRNA fragment we used a forward primer containing two inverted BsaI sites and a reverse primer containing a SalI site (Table S1). The two fragments were then ligated using an overlap extension PCR and ligated into pGEM/T‐Easy (Promega). Positive clones were digested with AscI and SalI, and the dropout was subsequently ligated into an AscI‐ and SalI‐linearized pENTR‐PpU6‐sgRNA‐NGG plasmid.

To generate the six entry vectors compatible with Multisite Gateway (Invitrogen) for multiplexing experiments, we amplified the sgRNA expression cassette from pENTR‐PpU6‐sgRNA‐L1L2 using primers (Table S1) containing different Multisite Gateway attachment sites (attB) and subsequently recombined with the Multisite Gateway pDONR221 plasmid set (Invitrogen) using a BP clonase reaction following the manufacturer's recommendations.