Development of microtiter plate scale CRISPR/Cas9 transformation method for Aspergillus niger based on in vitro assembled ribonucleoprotein complexes

Joosu Kuivanen (Corresponding Author), Veera Korja, Sami Holmström, Peter Richard

Research output: Contribution to journalArticleScientificpeer-review

5 Citations (Scopus)

Abstract

Background: The CRISPR/Cas9 is currently the predominant technology to enhance the genome editing efficiency in eukaryotes. Established tools for many fungal species exist while most of them are based on in vivo expressed Cas9 and guide RNA (gRNA). Alternatively, in vitro assembled Cas9 and gRNA ribonucleoprotein complexes can be used in genome editing, however, only a few examples have been reported in fungi. In general, high-throughput compatible transformation workflows for filamentous fungi are immature.

Results: In this study, a CRISPR/Cas9 facilitated transformation and genome editing method based on in vitro assembled ribonucleoprotein complexes was developed for the filamentous fungus Aspergillus niger. The method was downscaled to be compatible with 96-well microtiter plates. The optimized method resulted in 100% targeting efficiency for a single genomic target. After the optimization, the method was demonstrated to be suitable for multiplexed genome editing with two or three genomic targets in a metabolic engineering application. As a result, an A. niger strain with improved capacity to produce galactarate, a potential chemical building block, was generated.

Conclusions: The developed microtiter plate compatible CRISPR/Cas9 method provides a basis for high-throughput genome editing workflows in A. niger and other related species. In addition, it improves the cost-effectiveness of CRISPR/Cas9 genome editing methods in fungi based on in vitro assembled ribonucleoproteins. The demonstrated metabolic engineering example with multiplexed genome editing highlights the applicability of the method.

Original languageEnglish
Article number3
Number of pages12
JournalFungal Biology and Biotechnology
Volume6
Issue number3
DOIs
Publication statusPublished - 15 Mar 2019
MoE publication typeNot Eligible

Keywords

  • CRISPR
  • genome editing
  • high-throughput
  • automation
  • aspergillus niger
  • galactarate
  • mucic acid
  • pectin
  • galacturonate
  • metabolic engineering

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