Limits...
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.


Screening by PCR and sequencing. Expected sgRNA cut indicated by ↓. Red text shows the sgRNA target sequence and bold text the PAM motif. Primary clones: Several primary clones contain sequences showing both CRISPR-induced mutations and the wildtype (WT) sequence, as seen by the presence of two bands following PCR, these are indicated by (+WT). Mutants M1–M4 were selected on plates and LM1 underwent selection in liquid media. M1 shows a 4 nt deletion from the second sgRNA whilst mutants M2–M4 show a 37 nt deletion between the two CRISPR-Cas cut sites. Sub-clones: The gel shows examples of a selection of sub-clones derived from the primary clones. Sub-clones are labelled according to the primary clone and sub-clone number. With the exception of M1_9, which gives a WT sequence and 4 nt deletion as seen in the primary clone, all sub-clones chosen for sequencing are bi-allelic. Two-thirds of sequenced bi-allelic sub-clones show a single sequence with a 37 nt deletion suggesting that both alleles carry the same mutation. In sub-clones where mutations differ between alleles both sequences are shown
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC5121945&req=5

Fig2: Screening by PCR and sequencing. Expected sgRNA cut indicated by ↓. Red text shows the sgRNA target sequence and bold text the PAM motif. Primary clones: Several primary clones contain sequences showing both CRISPR-induced mutations and the wildtype (WT) sequence, as seen by the presence of two bands following PCR, these are indicated by (+WT). Mutants M1–M4 were selected on plates and LM1 underwent selection in liquid media. M1 shows a 4 nt deletion from the second sgRNA whilst mutants M2–M4 show a 37 nt deletion between the two CRISPR-Cas cut sites. Sub-clones: The gel shows examples of a selection of sub-clones derived from the primary clones. Sub-clones are labelled according to the primary clone and sub-clone number. With the exception of M1_9, which gives a WT sequence and 4 nt deletion as seen in the primary clone, all sub-clones chosen for sequencing are bi-allelic. Two-thirds of sequenced bi-allelic sub-clones show a single sequence with a 37 nt deletion suggesting that both alleles carry the same mutation. In sub-clones where mutations differ between alleles both sequences are shown

Mentions: The two CRISPR-Cas targets with the highest on target scores (0.5 and 0.79), containing a predicted cut site over a restriction site and occurring early in the coding region, were chosen. sgRNAs were designed to cut 37 nt apart at positions 138 and 175 within the urease gene. Both targets started with a G for polymerase III transcription (Fig. 2). No off-target sites were predicted for sgRNAs designed for either of the two CRISPR-Cas target sequences.Fig. 2


Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana
Screening by PCR and sequencing. Expected sgRNA cut indicated by ↓. Red text shows the sgRNA target sequence and bold text the PAM motif. Primary clones: Several primary clones contain sequences showing both CRISPR-induced mutations and the wildtype (WT) sequence, as seen by the presence of two bands following PCR, these are indicated by (+WT). Mutants M1–M4 were selected on plates and LM1 underwent selection in liquid media. M1 shows a 4 nt deletion from the second sgRNA whilst mutants M2–M4 show a 37 nt deletion between the two CRISPR-Cas cut sites. Sub-clones: The gel shows examples of a selection of sub-clones derived from the primary clones. Sub-clones are labelled according to the primary clone and sub-clone number. With the exception of M1_9, which gives a WT sequence and 4 nt deletion as seen in the primary clone, all sub-clones chosen for sequencing are bi-allelic. Two-thirds of sequenced bi-allelic sub-clones show a single sequence with a 37 nt deletion suggesting that both alleles carry the same mutation. In sub-clones where mutations differ between alleles both sequences are shown
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5121945&req=5

Fig2: Screening by PCR and sequencing. Expected sgRNA cut indicated by ↓. Red text shows the sgRNA target sequence and bold text the PAM motif. Primary clones: Several primary clones contain sequences showing both CRISPR-induced mutations and the wildtype (WT) sequence, as seen by the presence of two bands following PCR, these are indicated by (+WT). Mutants M1–M4 were selected on plates and LM1 underwent selection in liquid media. M1 shows a 4 nt deletion from the second sgRNA whilst mutants M2–M4 show a 37 nt deletion between the two CRISPR-Cas cut sites. Sub-clones: The gel shows examples of a selection of sub-clones derived from the primary clones. Sub-clones are labelled according to the primary clone and sub-clone number. With the exception of M1_9, which gives a WT sequence and 4 nt deletion as seen in the primary clone, all sub-clones chosen for sequencing are bi-allelic. Two-thirds of sequenced bi-allelic sub-clones show a single sequence with a 37 nt deletion suggesting that both alleles carry the same mutation. In sub-clones where mutations differ between alleles both sequences are shown
Mentions: The two CRISPR-Cas targets with the highest on target scores (0.5 and 0.79), containing a predicted cut site over a restriction site and occurring early in the coding region, were chosen. sgRNAs were designed to cut 37 nt apart at positions 138 and 175 within the urease gene. Both targets started with a G for polymerase III transcription (Fig. 2). No off-target sites were predicted for sgRNAs designed for either of the two CRISPR-Cas target sequences.Fig. 2

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.