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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.


PCR of the targeted urease fragment following growth of WT and mutant cell lines in nitrate or urea. NEB 100 bp ladder (1), WT in nitrate (2) and urea (3), M2_10 in nitrate (4) and urea (5) and M3_9 in nitrate (6) and urea (7)
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Fig5: PCR of the targeted urease fragment following growth of WT and mutant cell lines in nitrate or urea. NEB 100 bp ladder (1), WT in nitrate (2) and urea (3), M2_10 in nitrate (4) and urea (5) and M3_9 in nitrate (6) and urea (7)

Mentions: Four putative bi-allelic mutants (LM1, M4, M2_10 and M3_9) were tested along with WT and the mono-allelic M1_10 over two growth curve experiments. Both LM1 from liquid selection (p = 0.0029) and the sub-clone M3_9 (p = 0.0000001) showed a significant decrease in growth rate in urea compared to nitrate (Fig. 3) as well as a significant 13–18% decrease in cell size (Fig. 4; p = 0.0029 and p = 0, respectively). The latter was also apparent with light microscopy (results not shown). Mutants in urea could be easily discerned even without cell counts, as cultures appeared much paler in colour. M4 did not show a difference in growth rate but did show a significant decrease in cell size (p = 0.038).The mono-allelic mutant M1_10, displayed higher growth in urea and similar growth to the WT control (Fig. 3). This correlates with results from Weyman et al. [17] which showed that despite a reduced protein concentration, a mono-allelic urease knock-out was able to grow in urea. M2_10 which screened as a bi-allelic mutant prior to growth experiments showed a smaller but still significant decrease in growth rate (p = 0.0014; Fig. 3) and cell size (p = 0.0039; Fig. 4). PCR screening of the urease gene following growth in nitrate and urea showed the expected bi-allelic mutation for LM1, M3_9 and M4, however M2_10 also showed a faint WT band in nitrate and a strong WT band in urea (Fig. 5). This suggests that M2_10 was mosaic, with cells containing a functional urease out-competing those with a mutant urease. Given that only a faint WT band was present after growth in nitrate this suggests that the majority of the cells from the sub clone contained the mutant urease, initially accounting for the majority of growth and resulting in a lower but still significant decrease in growth rate.Fig. 3


Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana
PCR of the targeted urease fragment following growth of WT and mutant cell lines in nitrate or urea. NEB 100 bp ladder (1), WT in nitrate (2) and urea (3), M2_10 in nitrate (4) and urea (5) and M3_9 in nitrate (6) and urea (7)
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5121945&req=5

Fig5: PCR of the targeted urease fragment following growth of WT and mutant cell lines in nitrate or urea. NEB 100 bp ladder (1), WT in nitrate (2) and urea (3), M2_10 in nitrate (4) and urea (5) and M3_9 in nitrate (6) and urea (7)
Mentions: Four putative bi-allelic mutants (LM1, M4, M2_10 and M3_9) were tested along with WT and the mono-allelic M1_10 over two growth curve experiments. Both LM1 from liquid selection (p = 0.0029) and the sub-clone M3_9 (p = 0.0000001) showed a significant decrease in growth rate in urea compared to nitrate (Fig. 3) as well as a significant 13–18% decrease in cell size (Fig. 4; p = 0.0029 and p = 0, respectively). The latter was also apparent with light microscopy (results not shown). Mutants in urea could be easily discerned even without cell counts, as cultures appeared much paler in colour. M4 did not show a difference in growth rate but did show a significant decrease in cell size (p = 0.038).The mono-allelic mutant M1_10, displayed higher growth in urea and similar growth to the WT control (Fig. 3). This correlates with results from Weyman et al. [17] which showed that despite a reduced protein concentration, a mono-allelic urease knock-out was able to grow in urea. M2_10 which screened as a bi-allelic mutant prior to growth experiments showed a smaller but still significant decrease in growth rate (p = 0.0014; Fig. 3) and cell size (p = 0.0039; Fig. 4). PCR screening of the urease gene following growth in nitrate and urea showed the expected bi-allelic mutation for LM1, M3_9 and M4, however M2_10 also showed a faint WT band in nitrate and a strong WT band in urea (Fig. 5). This suggests that M2_10 was mosaic, with cells containing a functional urease out-competing those with a mutant urease. Given that only a faint WT band was present after growth in nitrate this suggests that the majority of the cells from the sub clone contained the mutant urease, initially accounting for the majority of growth and resulting in a lower but still significant decrease in growth rate.Fig. 3

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.