Fig. 1

Single-locus integration efficiency of gRNA targets in P. pastoris Δku70 strain. a Schematic illustration of the DSB introduced by Cas9 cleavage, which is repaired predominantly by homology directed repair (HDR) in Δku70, but by non-homologous-end-joining (NHEJ) in wild type. b Schematic illustration of gRNA targets selected within 100-bp upstream of TEF1-α, FLD1, AOX1, and GAP promoters and downstream of AOX1 terminator. Potential gRNA binding sequences were confirmed by navigating PAM sequence and assessed with an online software CHOPCHOP, which finally suggested 10 highly scored sequences (marked by green arrows) distributed on different chromosomes (Table 1). The eGFP expression cassette flanked by 1000-bp homologous arms was used for single-locus integration efficiency analysis (Additional file 1: Fig. S2). Five efficient gRNA targets located in different regions were selected for subsequent gene integration experiments. c Targeting efficiencies and CFUs of the selected 5 gRNA targets. Transformants of each gRNA target were separately picked and cultured in YNDH medium. The eGFP fluorescence intensity was measured at 72 h (Additional file 1: Fig. S3). Three highly efficient gRNA targets (P_AOX1UP-g2, P_TEF1UP-g1, and P_FLD1UP-g1) were then selected for subsequent multiloci integration experiments