Fig. 2

CRISPR–Cas9-mediated multiloci integration in P. pastoris Δku70 strain. a Overview of double- and triple-locus integration. Three fluorescent proteins were used as reporter for efficiency analysis of double-locus (DLI, eGFP and mCherry) and triple-locus (TLI, eGFP, mCherry and BFP) integration. The gRNA coding sequence and its self-cleaving ribozymes flanking both sides constitute the RGR operon. Multiple tandem RGR operon and codon optimized CAS gene were co-regulated by the bidirectional promoter P_HTX1. The RGR operon shown in the dashed box represents TLI. b Targeting efficiencies and CFUs of DLI. Similar to single-locus integration experiments, the transformants with abnormal fluorescence intensity were excluded from analysis (Additional file 1: Fig. S4). The integration efficiency of the four experimental groups ranged between 57.7 and 70%. The combination of P_FLD1UP-g1 and P_TEF1UP-g1 resulted in fewer colonies than that of P_TEF1UP-g1 and P_AOX1UP-g2. c Targeting efficiencies and CFUs of TLI. As with previous experiments, only transformants that normally expressed three fluorescent proteins were counted (Additional file 1: Fig. S5). The CFUs and integration efficiency of TLI were relatively low for all six experimental groups independent of the donor DNAs