Dataset Information

Efficient genetic transformation and CRISPR/Cas9-mediated genome editing in Lemna aequinoctialis.

ABSTRACT: The fast growth, ease of metabolic labelling and potential for feedstock and biofuels production make duckweeds not only an attractive model system for understanding plant biology, but also a potential future crop. However, current duckweed research is constrained by the lack of efficient genetic manipulation tools. Here, we report a case study on genome editing in a duckweed species, Lemna aequinoctialis, using a fast and efficient transformation and CRISPR/Cas9 tool. By optimizing currently available transformation protocols, we reduced the duration time of Agrobacterium-mediated transformation to 5-6 weeks with a success rate of over 94%. Based on the optimized transformation protocol, we generated 15 (14.3% success rate) biallelic LaPDS mutants that showed albino phenotype using a CRISPR/Cas9 system. Investigations on CRISPR/Cas9-mediated mutation spectrum among mutated L. aequinoctialis showed that most of mutations were short insertions and deletions. This study presents the first example of CRISPR/Cas9-mediated genome editing in duckweeds, which will open new research avenues in using duckweeds for both basic and applied research.

SUBMITTER: Liu Y

PROVIDER: S-EPMC6790374 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Efficient genetic transformation and CRISPR/Cas9-mediated genome editing in Lemna aequinoctialis.

Liu Yu Y Wang Yu Y Xu Shuqing S Tang Xianfeng X Zhao Jinshan J Yu Changjiang C He Guo G Xu Hua H Wang Shumin S Tang Yali Y Fu Chunxiang C Ma Yubin Y Zhou Gongke G

Plant biotechnology journal 20190503 11

The fast growth, ease of metabolic labelling and potential for feedstock and biofuels production make duckweeds not only an attractive model system for understanding plant biology, but also a potential future crop. However, current duckweed research is constrained by the lack of efficient genetic manipulation tools. Here, we report a case study on genome editing in a duckweed species, Lemna aequinoctialis, using a fast and efficient transformation and CRISPR/Cas9 tool. By optimizing currently av ...[more]

PMID: 30972865

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Project description:Genetic and molecular modifications of the large dsDNA chloroviruses, with genomes of 290 to 370 kb, would expedite studies to elucidate the functions of both identified and unidentified virus-encoded proteins. These plaque-forming viruses replicate in certain unicellular, eukaryotic chlorella-like green algae. However, to date, only a few of these algal species and virtually none of their viruses have been genetically manipulated due to lack of practical methods for genetic transformation and genome editing. Attempts at using Agrobacterium-mediated transfection of chlorovirus host Chlorella variabilis NC64A with a specially-designed binary vector resulted in successful transgenic cell selection based on expression of a hygromycin-resistance gene, initial expression of a green fluorescence gene and demonstration of integration of Agrobacterium T-DNA. However, expression of the integrated genes was soon lost. To develop gene editing tools for modifying specific chlorovirus CA-4B genes using preassembled Cas9 protein-sgRNA ribonucleoproteins (RNPs), we tested multiple methods for delivery of Cas9/sgRNA RNP complexes into infected cells including cell wall-degrading enzymes, electroporation, silicon carbide (SiC) whiskers, and cell-penetrating peptides (CPPs). In one experiment two independent virus mutants were isolated from macerozyme-treated NC64A cells incubated with Cas9/sgRNA RNPs targeting virus CA-4B-encoded gene 034r, which encodes a glycosyltransferase. Analysis of DNA sequences from the two mutant viruses showed highly targeted nucleotide sequence modifications in the 034r gene of each virus that were fully consistent with Cas9/RNP-directed gene editing. However, in ten subsequent experiments, we were unable to duplicate these results and therefore unable to achieve a reliable system to genetically edit chloroviruses. Nonetheless, these observations provide strong initial suggestions that Cas9/RNPs may function to promote editing of the chlorovirus genome, and that further experimentation is warranted and worthwhile.

Dataset Information

Efficient genetic transformation and CRISPR/Cas9-mediated genome editing in Lemna aequinoctialis.

Publications

Efficient genetic transformation and CRISPR/Cas9-mediated genome editing in Lemna aequinoctialis.

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