Expanding the CRISPR/Cas9 toolkit for Pichia pastoris with efficient donor integration and alternative resistance markers

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60461373%3A22330%2F18%3A43917110" target="_blank" >RIV/60461373:22330/18:43917110 - isvavai.cz</a>

Result on the web

<a href="https://onlinelibrary.wiley.com/doi/pdf/10.1002/jcb.26474" target="_blank" >https://onlinelibrary.wiley.com/doi/pdf/10.1002/jcb.26474</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1002/jcb.26474" target="_blank" >10.1002/jcb.26474</a>

Result language

angličtina

Original language name

Expanding the CRISPR/Cas9 toolkit for Pichia pastoris with efficient donor integration and alternative resistance markers

Original language description

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. © 2017 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.

Czech name

—

Czech description

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Type

J_imp - Article in a specialist periodical, which is included in the Web of Science database

CEP classification

—

OECD FORD branch

10608 - Biochemistry and molecular biology

Project

—

Continuities

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Publication year

2018

Confidentiality

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Name of the periodical

Journal of Cellular Biochemistry

ISSN

0730-2312

e-ISSN

—

Volume of the periodical

119

Issue of the periodical within the volume

4

Country of publishing house

US - UNITED STATES

Number of pages

16

Pages from-to

3183-3198

UT code for WoS article

000426187500019

EID of the result in the Scopus database

2-s2.0-85039166549