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Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana

View Article: PubMed Central - PubMed

ABSTRACT

Background: CRISPR-Cas is a recent and powerful addition to the molecular toolbox which allows programmable genome editing. It has been used to modify genes in a wide variety of organisms, but only two alga to date. Here we present a methodology to edit the genome of Thalassiosira pseudonana, a model centric diatom with both ecological significance and high biotechnological potential, using CRISPR-Cas.

Results: A single construct was assembled using Golden Gate cloning. Two sgRNAs were used to introduce a precise 37 nt deletion early in the coding region of the urease gene. A high percentage of bi-allelic mutations (≤61.5%) were observed in clones with the CRISPR-Cas construct. Growth of bi-allelic mutants in urea led to a significant reduction in growth rate and cell size compared to growth in nitrate.

Conclusions: CRISPR-Cas can precisely and efficiently edit the genome of T. pseudonana. The use of Golden Gate cloning to assemble CRISPR-Cas constructs gives additional flexibility to the CRISPR-Cas method and facilitates modifications to target alternative genes or species.

Electronic supplementary material: The online version of this article (doi:10.1186/s13007-016-0148-0) contains supplementary material, which is available to authorized users.

No MeSH data available.


Translated WT urease (a), frame 3 (b) and frame 1 of urease with the expected 37 nt deletion (c) and frame 1 of urease with a 38 nt deletion (d). Position of deletion indicated by ↓. The model WT protein contains 807aa. The figure shows the initial 260 amino acids for a and b including the start of the alpha sub-unit. Translations are identical for the unshown segments. Gamma (pink), Beta (green) and Alpha (blue) sub-units are highlighted in order. Expected start codon (red) and upstream out-of-frame start codons (grey) are highlighted
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Fig6: Translated WT urease (a), frame 3 (b) and frame 1 of urease with the expected 37 nt deletion (c) and frame 1 of urease with a 38 nt deletion (d). Position of deletion indicated by ↓. The model WT protein contains 807aa. The figure shows the initial 260 amino acids for a and b including the start of the alpha sub-unit. Translations are identical for the unshown segments. Gamma (pink), Beta (green) and Alpha (blue) sub-units are highlighted in order. Expected start codon (red) and upstream out-of-frame start codons (grey) are highlighted

Mentions: Translations of urease sequences with both 37 and 38 nt deletions show frame shifts and early stop codons after the deletion in the gamma sub-unit, leading to major disruption of the gamma sub-unit, nonsense down-stream and short products of 24 or 44 amino acid residues (Fig. 6). Since all mono-clonal bi-allelic mutants tested for growth in urea had either two alleles with a 37 nt deletion or both a 37 and 38 nt deletion, it was predicted that the urease gene would no longer be functional. However, several mechanisms exist in eukaryotes which can allow translation of the protein from start codons later in the coding region. These include leaky initiation, re-initiation of ribosomes and internal ribosome entry sites (IRES) [50]. IRES have been shown to become active in yeast following amino acid starvation [50]. If an in-frame translation can occur after the deletion at an IRES or via a mechanism such as re-initiation then the active site located in the alpha-subunit could still be present. The first in-frame ATG after the deletion would start translation of the protein just before the beta sub-unit, leading to an N-terminal truncated protein without the gamma sub-unit but with both the beta and alpha sub-units (Fig. 6). Earlier start codons are predicted to result in non-sense and early stop codons.Fig. 6


Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana
Translated WT urease (a), frame 3 (b) and frame 1 of urease with the expected 37 nt deletion (c) and frame 1 of urease with a 38 nt deletion (d). Position of deletion indicated by ↓. The model WT protein contains 807aa. The figure shows the initial 260 amino acids for a and b including the start of the alpha sub-unit. Translations are identical for the unshown segments. Gamma (pink), Beta (green) and Alpha (blue) sub-units are highlighted in order. Expected start codon (red) and upstream out-of-frame start codons (grey) are highlighted
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Related In: Results  -  Collection

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Fig6: Translated WT urease (a), frame 3 (b) and frame 1 of urease with the expected 37 nt deletion (c) and frame 1 of urease with a 38 nt deletion (d). Position of deletion indicated by ↓. The model WT protein contains 807aa. The figure shows the initial 260 amino acids for a and b including the start of the alpha sub-unit. Translations are identical for the unshown segments. Gamma (pink), Beta (green) and Alpha (blue) sub-units are highlighted in order. Expected start codon (red) and upstream out-of-frame start codons (grey) are highlighted
Mentions: Translations of urease sequences with both 37 and 38 nt deletions show frame shifts and early stop codons after the deletion in the gamma sub-unit, leading to major disruption of the gamma sub-unit, nonsense down-stream and short products of 24 or 44 amino acid residues (Fig. 6). Since all mono-clonal bi-allelic mutants tested for growth in urea had either two alleles with a 37 nt deletion or both a 37 and 38 nt deletion, it was predicted that the urease gene would no longer be functional. However, several mechanisms exist in eukaryotes which can allow translation of the protein from start codons later in the coding region. These include leaky initiation, re-initiation of ribosomes and internal ribosome entry sites (IRES) [50]. IRES have been shown to become active in yeast following amino acid starvation [50]. If an in-frame translation can occur after the deletion at an IRES or via a mechanism such as re-initiation then the active site located in the alpha-subunit could still be present. The first in-frame ATG after the deletion would start translation of the protein just before the beta sub-unit, leading to an N-terminal truncated protein without the gamma sub-unit but with both the beta and alpha sub-units (Fig. 6). Earlier start codons are predicted to result in non-sense and early stop codons.Fig. 6

View Article: PubMed Central - PubMed

ABSTRACT

Background: CRISPR-Cas is a recent and powerful addition to the molecular toolbox which allows programmable genome editing. It has been used to modify genes in a wide variety of organisms, but only two alga to date. Here we present a methodology to edit the genome of Thalassiosira pseudonana, a model centric diatom with both ecological significance and high biotechnological potential, using CRISPR-Cas.

Results: A single construct was assembled using Golden Gate cloning. Two sgRNAs were used to introduce a precise 37 nt deletion early in the coding region of the urease gene. A high percentage of bi-allelic mutations (≤61.5%) were observed in clones with the CRISPR-Cas construct. Growth of bi-allelic mutants in urea led to a significant reduction in growth rate and cell size compared to growth in nitrate.

Conclusions: CRISPR-Cas can precisely and efficiently edit the genome of T. pseudonana. The use of Golden Gate cloning to assemble CRISPR-Cas constructs gives additional flexibility to the CRISPR-Cas method and facilitates modifications to target alternative genes or species.

Electronic supplementary material: The online version of this article (doi:10.1186/s13007-016-0148-0) contains supplementary material, which is available to authorized users.

No MeSH data available.