CRISPR/Cas9 und Zinkfinger-Nukleasen für die gezielte Genstilllegung in Chlamydomonas reinhardtii

Die einzellige Grünalge Chlamydomonas reinhardtii ist ein vielseitiger Modellorganismus sowohl in der Grundlagenforschung als auch für biotechnologische Anwendung. Für die genetische Veränderung wurden verschiedene Methoden entwickelt, jedoch ist die gezielte Modifikation kerncodierter Gene immernoch sehr schwierig. In dieser Arbeit wird eine Strategie vorgestellt, die es ermöglicht, kerncodierte Gene in Chlamydomonas gezielt mit sequenzspezifischen Zinkfinger-Nukleasen zu verändern. Das COP3-Gen, welches den lichtaktivierbaren Ionenkanal Kanalrhodopsin-1 codiert, diente hierbei als Zielsequenz der für die Deletion hergestellten Zinkfinger-Nukleasen. Um eine Charakterisierung der ZFNs zu ermöglichen, wurde ein Modelstamm generiert, der ein inaktiviertes Markergen enthält. Die Inaktiverung erfolgte hierbei durch Insertion der COP3-ZFN Zielsequenz. Die Transformation dieses Modellstamms mit ZFN codierender Plasmid-DNA und einem Reparatur-Template ermöglichte die Wiederherstellung der Markeraktivität und eine Selektion Antibiotika-resistenter Kolonien. Wenn in diesen Experimenten zusätzlich ein COP3 veränderndes Template benutzt wurde, enthielt 1% der analysierten Klone ein mutiertes COP3-Gen. Der Chlamydomonas Augenfleck ist ein lichtsensitives Organell mit entscheidender Funktion für die phototaktische Orientierung der Alge. Eine Deletionsmutante des Blaulicht-Photorezeptors Phototropin zeigte in Experimenten eine veränderte Regulation der lichtabhängigen Augenfleckgröße. Durch Komplementierung der Phototropin-Dysfunktion konnte der lichtabhängige Regulationsprozess wiederhergestellt werden. Die Expression der Phototropin-Kinasedomäne führte zu einer lichtunabhängigen Reduktion der Augenfleckfläche. Interessanterweise führte auch die Expression der N-terminalen LOV-Domänen zu einer geänderten Regulation des Augenflecks und der Phototaxis. Dies deutet, zusätzlich zur Lichtregulation der Kinasedomäne, auf eine zelluläre Signalfunktion der LOV-Domänen hin.The unicellular green alga Chlamydomonas reinhardtii is a versatile model for fundamental and biotechnological research. A wide toolset for genetic manipulation has been developed for this alga, but specific modification of nuclear genes is still not routinely possible. Here we present a nuclear gene targeting strategy for Chlamydomonas that is based on the application of zinc-finger nucleases (ZFNs). Initially, we designed a set of ZFNs for targeting the COP3 gene that encodes the light-activated ion channel channelrhodopsin-1. To evaluate the designed ZFNs, we constructed a model strain by inserting a non-functional selection marker interspaced with a short COP3 target sequence into the nuclear genome. Upon co-transformation of this recipient strain with the engineered ZFNs and a DNA repair template, we were able to restore marker activity and select antibiotic resistant clones with active nucleases. In cases where cells were co-transformed with a modified COP3 template, 1% of these clones contained a modified COP3 locus as well. The eyespot of Chlamydomonas is a light-sensitive organelle important for phototactic orientation of the alga. Here we found that eyespot size is downregulated in light. In a strain in which the blue light photoreceptor phototropin was deleted, the light regulation of the eyespot size was affected. We restored this dysfunction in different phototropin complementation experiments. Complementation with the phototropin kinase fragment reduced the eyespot size, independent of light. Interestingly, overexpression of the N-terminal LOV-domains alone also affected eyespot size and phototaxis, suggesting that aside from activation of the kinase domain, they fulfill an independent signaling function in the cell. We propose that phototropin is a light regulator of phototaxis that desensitizes the eyespot when blue light intensities increase

Median-joining network showing genetic structure of the five species of bathymodiolin mussels from the GoM (<i>Bathymodiolus heckerae</i>, <i>B. childressi</i>, <i>B. brooksi</i>, <i>B</i>. nov. sp. GoM and <i>Tamu fisheri</i>) (CO1 data only).

<p>Size of the haplotype circle are proportional to their frequencies (Unique (n = 1); rare (n = 2 to 4), common (n = 5 to 20) and abundant (n = 20 to 28). Black points represent mutations. Diamonds represent missing haplotype. Colours represent locations were the haplotype was found. In the legend, the dashed line represents the virtual limit between “shallow population (less than or about 1000m deep) and deep populations (more than 1400m deep). The number of sequences used for each species and population is indicated on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118460#pone.0118460.t001" target="_blank">Table 1</a>.</p

Distribution of pairwise K2P distance-frequency histogram from CO1 and ND4 concatenated data.

<p>Distribution of pairwise K2P distance-frequency histogram from CO1 and ND4 concatenated data.</p

<i>B. heckerae</i> site-specific genetic diversity.

<p><i>N</i>: number of individuals</p><p><i>h</i>: number of haplotypes</p><p><i>Hd</i>: Haplotype diversity</p><p><i>S</i>: number of segregating site</p><p>π: Average number of nucleotide differences per site between two sequences [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118460#pone.0118460.ref034" target="_blank">34</a>]</p><p><i>B. heckerae</i> site-specific genetic diversity.</p

Estimated time of divergence between GoM and Atlantic <i>Bathymodiolus</i> species (in MY).

<p>Time of divergence is estimated using the CO1 mutation rate of 0.39% / MY and divergence values from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118460#pone.0118460.t008" target="_blank">Table 8</a>.</p

Summary statistics for mitochondrial loci polymorphism.

<p>Locus: CO1 (545bp)</p><p>ND4 (678bp)</p><p>concatenate C01+ND4: 1123bp)</p><p><i>N</i>: number of sequences</p><p><i>h</i>: number of haplotypes</p><p><i>Hd</i>: haplotype diversity</p><p><i>S</i>: Number of segregating sites</p><p><i>π</i>: nucleotide diversity</p><p>Θ_<i>w</i>: Watterson’s Theta per site from S</p><p>Tajima’s D: Tajima’s statistic [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118460#pone.0118460.ref035" target="_blank">35</a>]</p><p>Statistical significance</p><p>*,P < 0.05</p><p>**, P < 0.01</p><p>***, P < 0.001</p><p>Summary statistics for mitochondrial loci polymorphism.</p

Map of collection sites in the Gulf of Mexico.

<p>Sampling effort, depth and coordinates are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118460#pone.0118460.t001" target="_blank">Table 1</a>.</p

CO1 divergence between GoM and Atlantic Bathymodiolus species.

<p>Divergence is estimated as the average number of nucleotidic differences per site between populations.</p

Assignment of species after genetic identification (as percentage of total).

<p>Percentage of assignments after removing individuals of the new species from site DC583 from the calculation are shown in parentheses.</p><p>Assignment of species after genetic identification (as percentage of total).</p

<i>B. heckerae</i> ɸ_st population differentiation, tested by 1023 permutations with the Arlequin software for the concatenated CO1 and ND4 genes.

<p><i>B. heckerae</i> ɸ_st population differentiation, tested by 1023 permutations with the Arlequin software for the concatenated CO1 and ND4 genes.</p