Px330 spcas9 ng

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)).

To construct pEX-FlagR-ABEmax and pEX-FlagR-BE4max, the ABEmax and BE4max coding sequences (CDSs) were amplified from pCMV_ABEmax (Addgene no.112095) and pCMV_AncBE4max (Addgene no. 112094), respectively, and cloned into the mammalian expression pEX-FlagR vector using the Xho I and Xba I restriction sites. To construct pCMV-NGABEmax, we replaced the Cas9 sequence in pCMV_ABEmax with the SpCas9-NG sequence from pX330-SpCas9-NG (Addgene no. 117919) using the Pml I and Eco RI restriction sites. To construct pEX-FlagR-NGABEmax, the NGABEmax sequence from pCMV-NGABEmax was cloned into pEX-FlagR. Gibson fragments containing the ABEmax or BE4max CDS with matching overlaps were polymerase chain reaction (PCR)–amplified using Phusion High-Fidelity DNA Polymerase [New England Biolabs (NEB)]. Fragments were gel-purified and assembled using NEBuilder HiFi DNA Assembly master mix (NEB) for 1 hour at 50°C and transformed into chemically competent E. coli (DH5α; Enzynomics). Sequences corresponding to sgRNAs were cloned into Bsa I–digested pRG2 vector (Addgene no. 104174). For this step, oligos containing the spacer sequence (table S3) were annealed to form double-stranded DNA fragments with compatible overhangs and ligated using T4 ligase (Enzynomics). All plasmids used for transfection experiments were prepared using a NucleoBond Xtra Midi Plus EF kit [Macherey-Nagel (MN)].