Pgl3 u6 sgrna egfp

For construction of sgRNAs, oligos were synthesized, annealed, and cloned into BsaI site of the sgRNA expression vector. Plasmids used include pGL3-U6-sgRNA-PGK-puromycin (Addgene, 51133), pUC57-sgRNA expression vector (Addgene, 51132), pCMV-SaBE3 (Addgene, 85169), pGFP-N1 (Addgene, 54712), pUC57-Sa sgRNA expression vector, pGL3-U6-sgRNA-EGFP, pGL3-U6-sgRNA-BFP, and pCMV-ABE.

pCMV-PE2 plasmid was purchased from Addgene (Addgene, #132775). For expression of the WT pegRNA, the plasmid backbone was amplified from pGL3-U6-sgRNA-EGFP (Addgene, 107721) using Phanta® Max Super Fidelity DNA Polymerase (Vazyme) for removal of the original sgRNA scaffold (Supplementary Table 1). The resulting plasmid was cut by BsaI-HFv2 (NEB) for overhangs. The pegRNA scaffold oligos (featuring compatible overhangs) (Supplementary Table 1), spacer oligos (top strand with ends of 5′ ACCG and 3′ GTTTT, bottom strand with 5′ CTCTAAAAC overhang), and pegRNA 3′ extension oligos (top strand with the 5′ GTGC overhang, bottom strand with 5′ AAAA overhang) were synthesized and annealed. The annealed scaffold fragment was phosphorylated. Finally, four fragments (annealed spacer, annealed 3′ extension, phosphorylated scaffold, and the cut backbone) were assembled by DNA ligase. The sequence information for pegRNAs and nick-sgRNAs is provided in Supplementary Data 1. Assembled plasmids were transformed into E. coli and screened using Ampicillin. Based on the pegRNA construct, the joining of xrRNA sequence was carried out using recombinase-based cloning (Vazyme, ClonExpress II One Step Cloning Kit, #C112-02-AB).