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Construction of a series of episomal plasmids and their application in the development of an efficient CRISPR/Cas9 system in Pichia pastoris
- Source :
- World Journal of Microbiology and Biotechnology. 35
- Publication Year :
- 2019
- Publisher :
- Springer Science and Business Media LLC, 2019.
-
Abstract
- The methylotrophic yeast Pichia pastoris is widely used in recombinant expression of eukaryotic proteins owing to the ability of post-translational modification, tightly regulated promoters, and high cell density fermentation. However, episomal plasmids for heterologous gene expression and the CRISPR/Cas9 system for genome editing have not been well developed in P. pastoris. In the present study, a panel of episomal plasmids containing various autonomously replicating sequences (ARSs) were constructed and their performance in transformation efficiency, copy numbers, and propagation stability were systematically compared. Among the five ARSs with different origins, panARS isolated from Kluyveromyces lactis was determined to have the best performance and used to develop an efficient CRISPR/Cas9 based genome editing system. Compared with a previously reported system using the endogenous and most commonly used ARS (PARS1), the CRISPR/Cas9 genome editing efficiency was increased for more than tenfold. Owing to the higher plasmid stability with panARS, efficient CRISPR/Cas9-mediated genome editing with a type III promoter (i.e. SER promoter) to drive the expression of the single guide RNA (sgRNA) was achieved for the first time. The constructed episomal plasmids and developed CRISPR/Cas9 system will be important synthetic biology tools for both fundamental studies and industrial applications of P. pastoris.
- Subjects :
- DNA Replication
0106 biological sciences
Physiology
Autonomously replicating sequence
Genetic Vectors
Gene Dosage
Computational biology
01 natural sciences
Applied Microbiology and Biotechnology
Genomic Instability
Pichia
Pichia pastoris
Gene Knockout Techniques
Industrial Microbiology
Kluyveromyces
03 medical and health sciences
Synthetic biology
Transformation, Genetic
Plasmid
Genome editing
Gene Expression Regulation, Fungal
010608 biotechnology
Escherichia coli
CRISPR
Promoter Regions, Genetic
Gene Editing
Kluyveromyces lactis
0303 health sciences
biology
030306 microbiology
Cas9
General Medicine
biology.organism_classification
Synthetic Biology
CRISPR-Cas Systems
Genetic Engineering
Plasmids
RNA, Guide, Kinetoplastida
Biotechnology
Subjects
Details
- ISSN :
- 15730972 and 09593993
- Volume :
- 35
- Database :
- OpenAIRE
- Journal :
- World Journal of Microbiology and Biotechnology
- Accession number :
- edsair.doi.dedup.....cc971c17a3db81b0b97ab8dc39c13cc7