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DNA-free genome editing of Brassica oleracea and B. rapa protoplasts using CRISPR-Cas9 ribonucleoprotein complexes
Murovec, Jana (Author), Guček, Katja (Author), Bohanec, Borut (Author), Avbelj, Monika (Author), Jerala, Roman (Author)

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Abstract
The CRISPR/Cas9 genome editing system has already proved its efficiency, versatility and simplicity in numerous applications in human, animal, microbe and plant cells. Together with the vast amount of genome and transcriptome databases available, it represents an enormous potential for plant breeding and research. Although most changes produced with CRISPR/Cas9 do not differ from naturally occurring mutations, the use of transgenesis during varietal development can still trigger GMO legislation in countries that rely on process-based regulation. Moreover, stable integration of DNA coding for genome-editing tools into plant genomes can result in insertional mutagenesis, while its prolonged expression can cause mutations in off-target sites. These pitfalls can be avoided with the delivery of preassembled ribonucleoprotein complexes (RNPs) composed of purified recombinant enzyme Cas9 and in vitro-transcribed or synthesized sgRNA. We therefore aimed to develop a DNA-free protocol for site-directed mutagenesis of three species of the genus Brassica (B. oleracea, B. napus, and B. rapa) with the use of RNPs. We chose cabbage, rapeseed and Chinese cabbage as species representatives and introduced RNPs into their protoplasts with PEG 4000. Four sgRNAs targeting two endogenous genes (the FRI and PDS genes, two sgRNAs per gene) were introduced into all three species. No mutations were detected after transfection of rapeseed protoplasts, while we obtained mutation frequencies of 0.09 to 2.25% and 1.15 to 24.51% in cabbage and Chinese cabbage, respectively. In both species, a positive correlation was displayed between the amount (7.5, 15, 30, and 60 μg) of Cas9 enzyme and sgRNA introduced and mutation frequency. Nucleotide changes (insertions and deletions) were detected 24 h after transfection and did not differ 72 h after transfection. They were species-, gene- and locus-dependent. In summary, we demonstrated the suitability of RNP transfection into B. oleracea and B. rapa protoplasts for high-efficiency indel induction of two endogenous genes. Due to the relatively high mutation frequencies detected (up to 24.51%), this study paves the way for regeneration of precisely mutated Brassica plants without the use of transgenesis.

Language:English
Keywords:RGEN, CRISPR/Cas9, genome editing, NGS, ribonucleoproteins, protoplast, genus Brassica, B. napus
Work type:Article (dk_c)
Tipology:1.01 - Original Scientific Article
Organization:BF - Biotechnical Faculty
Year:2018
Number of pages:9 str.
Numbering:Vol. 9, art. 1594
UDC:577
ISSN on article:1664-462X
DOI:10.3389/fpls.2018.01594 This link opens in a new window
COBISS.SI-ID:9081209 This link opens in a new window
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Downloads:64
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Record is a part of a journal

Title:Frontiers in plant science
Shortened title:Front. plant sci.
Publisher:Frontiers Research Foundation
ISSN:1664-462X
COBISS.SI-ID:3011663 This link opens in a new window

Document is financed by a project

Funder:ARRS - Agencija za raziskovalno dejavnost Republike Slovenije (ARRS)
Project no.:P4-0077
Name:Kmetijske rastline - genetika in sodobne tehnologije

Funder:ARRS - Agencija za raziskovalno dejavnost Republike Slovenije (ARRS)
Project no.:P4-0176
Name:Molekularna biotehnologija: od dinamike bioloških sistemov do aplikacij

Funder:ARRS - Agencija za raziskovalno dejavnost Republike Slovenije (ARRS)
Project no.:J4-9307
Name:Preurejanje genomov izbranih vrst rodu Brassica s tehnologijo CRISPR/Cas9

Licences

License:CC BY 4.0, Creative Commons Attribution 4.0 International
Link:http://creativecommons.org/licenses/by/4.0/
Description:This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.
Licensing start date:05.11.2018

Secondary language

Language:Slovenian
Keywords:urejanje genoma, genetika

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