Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris
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- 作者：Astrid Weninger ; Anna-Maria Hatzl ; Christian Schmid ; Thomas Vogl ; thomas.vogl@aon.at" class="auth_mail" title="E-mail the corresponding author ; Anton Glieder
- 关键词：Genome engineering ; CRISPR/Cas9 ; Pichia pastoris ; Synthetic biology ; Gene replacement by homologous recombination ; Multiplexing
- 刊名：Journal of Biotechnology
- 出版年：2016
- 出版时间：10 October 2016
- 年：2016
- 卷：235
- 期：Complete
- 页码：139-149
- 全文大小：1850 K
文摘
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A CRISPR/Cas9 system has been developed for the methylotrophic yeast P. pastoris.
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Several parts for expression (promoters for CAS9 and gRNAs, CAS9 CDSs…) needed optimization.
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Only 6/95 constructs designed achieved high efficiency genome targeting.
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Targeting rates close to 100% and multiplexing were demonstrated for several genes.
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This report may serve as a guideline to implement CRISPR/Cas9 in other eukaryotes.