Abstract
Komagataella phaffii (syn. Pichia pastoris) is one of the most commonly used host systems for recombinant protein expression. Achieving targeted genetic modifications had been hindered by low frequencies of homologous recombination (HR). Recently, a CRISPR/Cas9 genome editing system has been implemented for P. pastoris enabling gene knockouts based on indels (insertion, deletions) via non‐homologous end joining (NHEJ) at near 100% efficiency. However, specifically integrating homologous donor cassettes via HR for replacement studies had proven difficult resulting at most in ∼20% correct integration using CRISPR/Cas9. Here, we demonstrate the CRISPR/Cas9 mediated integration of markerless donor cassettes at efficiencies approaching 100% using a ku70 deletion strain. The Ku70p is involved in NHEJ repair and lack of the protein appears to favor repair via HR near exclusively. While the absolute number of transformants in the Δku70 strain is reduced, virtually all surviving transformants showed correct integration. In the wildtype strain, markerless donor cassette integration was also improved up to 25‐fold by placing an autonomously replicating sequence (ARS) on the donor cassette. Alternative strategies for improving donor cassette integration using a Cas9 nickase variant or reducing off targeting associated toxicity using a high fidelity Cas9 variant were so far not successful in our hands in P. pastoris. Furthermore we provide Cas9/gRNA expression plasmids with a Geneticin resistance marker which proved to be versatile tools for marker recycling. The reported CRSIPR‐Cas9 tools can be applied for modifying existing production strains and also pave the way for markerless whole genome modification studies in P. pastoris.
Highlights
Within a few years, genome engineering by CRISPR/Cas[9] became one of the most valuable tools in biosciences.[1,2,3] This method facilitates targeted, programmable genome modifications and provides major advantages over classical knock-out, knockin-approaches as well as alternative genome engineering strategies
nonhomologous end joining (NHEJ)-repair appears strongly impaired using CRISPR/Cas[9] in P. pastoris Δku[70] strain As the CRISPR/Cas[9] mediated integration of donor cassettes in P. pastoris wildtype strains was so far inefficient and in part even unsuccessful,[32] we evaluated to combine the potency of the P. pastoris Δku[70] strain and CRISPR/Cas[9] to increase the homologous recombination (HR) rates and to enable the integration of markerless fragments
Interesting, we were not able to amplify the GUT1 locus of in Δgut[1] mutants with primers, which had been used for the amplification of genomic DNA isolated from P. pastoris wildtype CRISPR/ Cas[9] transformants (Figure 1D). These results strongly suggest that NHEJ is practically completed abolished in the Δku[70] strain: First, the transformation rates were drastically reduced compared to the wildtype strain (∼100-fold) when coexpressing gRNAs, indicating that double strand break (DSB) introduced by Cas[9] cannot be repaired and are lethal
Summary
Genome engineering by CRISPR/Cas[9] (clustered regularly interspaced short palindromic repeats/ CRISPR associated protein 9) became one of the most valuable tools in biosciences.[1,2,3] This method facilitates targeted, programmable genome modifications and provides major advantages over classical knock-out-, knockin-approaches as well as alternative genome engineering strategies. ARSs are typically placed on plasmids to allow episomal expression, which is beneficial for the production of selected proteins.[32] Depending on the ARS multiple copies of the plasmids are present in the cell (PARS1, 10 copies) and the plasmids are maintained in the presence of selective pressure.[33] Another previously reported strategy to increase the HR frequencies is the expression of the Cas[9] nickase, which was evaluated in this study This Cas[9] variant creates single strand breaks due to the inactivation of one of its catalytic domains via the introduction of a point mutation.[34,35] By introducing single strand breaks an increase in donor recombination was demonstrated by[36,37] in mammalian cells. The use of the Cas[9] nickase did so far not show clear advantages in our hands in P. pastoris
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