The Journal of Maltese Science – "Perfect Outcast" Edition

[Hunor]

The Journal of Maltese Science - "Perfect Outcast" Edition
30.12.828

Genetic principles for the further development of Cardamine
A paradigm shift in the use of Cardamine

Prof. P. Ferrari et al

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Introduction

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The present research results include the results of the Galicia Research Station Botanical and Biochemical Laboratory, which were born and achieved a breakthrough in the development of Cardamine. Over the past few years Cardamine has emerged as a powerful essential plant in the Outcast society, in foreign policy and the economy. Genetic studies have provided a powerful platform to identify the molecular causes of trait diversity between these species and for understanding the morphogenetic basis. As genome editing has opened considerable new potential for efforts to connect genotype to phenotype through development and evolution, it became important to set up a CRISPR/Cas9 genome editing in this system.

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Material and Methods

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To develop and evaluate a protocol for applying CRISPR/Cas9-mediated targeted mutagenesis in Cardamine, we selected its RCO gene. Two sgRNAs were designed to guide the SpCas9 endonuclease to the RCO locus in order to generate additional alleles with the potential to uncover new aspects of RCO function.

Figure 1. Schematic representation of the ChRCO locus targeted for mutagenesis. The two sgRNA complementary sequences are shown in red, and the PAM site is shown in blue. The yellow asterisk in exon 2 marks the position of the premature stop codon causing the first described rco mutant allele.

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Results

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To ensure the high expression of the SpCas9 protein in Cardamine, we focused on two known strong constitutive promoters: CaMV 35S (p35S) and ubiquitin. To get a first impression of how many heritable mutations sould be expected, genomic DNA wase xtracted from the primary inflorescences of randomly picked plants. Plants with 50% or more wild-type sequence traces are classified either as carrying targeted mutations with ‘low heredity probability’, or as ‘wild type’ if indels are present with less than 15%.

Phenotypic and genotypic analysis of the Cas9-free homozygous mutant progeny from two independent pPcUbi4-2::Cas9 lines, clearly differs from the wild type, e.g., much less dissected, but is virtually indistinguishable from those of the previously isolated rco mutant. Consistent with this, the new CRISPR mutants were found to be allelic to the original rco mutant allele in an allelism test. As expected, the respective genotypes lacked the T-DNA insertion containing Cas9, were homozygous for the mutant alleles. Mutations due to single nucleotide insertions in three independently CRISPR-derived alleles are indicated in lower case magenta. The previously known ‘non-CRISPR’ mutation in the second exon is shown in lower case green.

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Discussion and implication

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For the construct carrying p35S::SpCas9, such mutations could be detected only in ca. 40% of the T1 plants which, moreover, all had low probability of heredity, i.e., present with low frequency in reproductive tissue. In this respect, the codon-optimized Cas9 driven by the parsley PcUbi4-2 promoter clearly performed better. For the latter, all tested T1 plants carried targeted mutations with ca. 60% of these with low and ca. 40% with a high probability of heredity.
Genetic stability and increased expression showed the presence of significant active ingredient. The concentration of the active substance represented an average increase of 50% in the measured microenvironment. The stability of the molecule allows exploring the potential possibilities of different states and allows the human research phase of the candidate molecule. It is recommended to create an additional secured environment for the industrial cultivation of tested Cardamine varieties. In order to put the results of the present research into practice and to develop the new type of Cardamine, the scientific community of Malta recommends the development and construction of a subsurface and surface base on Planet Albegna. Further developments, investments and construction are needed.

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Background

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Orange Dream - Prof. Petra Ferrari
Orange Dream - Status Update - Prof. Petra Ferrari

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