Generating NG-PAM Targetable PE Vectors

To construct PE plasmid, PE cassette was amplified from pCMV-PE2 (Addgene no.132775) and amplified product was inserted into pBAtC (Addgene no.78097), generating pBAtC-NGG-PE2 vector. To build NG-PAM targetable PE vector, we introduced same mutations with pX330-SpCas9-NG (Addgene no.117919) in our Cas9 fragment. For introducing pegRNA cassette, oligos representing the target sequences, sgRNA scaffold and 3’ extensions were annealed and cloned into pRG2 vector (Addgene no. 104174) with additional AtU6-26 promoter using BsaI to build AtU6-26p-pegRNA vector. Restriction enzyme-digested fragment encoding AtU6-26p-pegRNA cassette was inserted into pBAtC-NG-PE2 vector digested with same restriction enzyme. To construct nicking sgRNA cassette, oligos representing nicking sequences were annealed and cloned into AarI-digested PE plasmid. Oligos used for preparing plasmid was designed using Cas-designer (22 (link)) and PE-designer (23 (link)).