Edvardsen Rolf B, Leininger Sven, Kleppe Lene, Skaftnesmo Kai Ove, Wargelius Anna
Institute of Marine Research, Bergen, Norway.
PLoS One. 2014 Sep 25;9(9):e108622. doi: 10.1371/journal.pone.0108622. eCollection 2014.
Understanding the biological function behind key proteins is of great concern in Atlantic salmon, both due to a high commercial importance and an interesting life history. Until recently, functional studies in salmonids appeared to be difficult. However, the recent discovery of targeted mutagenesis using the CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) system enables performing functional studies in Atlantic salmon to a great extent. We used the CRISPR/Cas9 system to target two genes involved in pigmentation, tyrosinase (tyr) and solute carrier family 45, member 2 (slc45a2). Embryos were assayed for mutation rates at the 17 somite stage, where 40 and 22% of all injected embryos showed a high degree of mutation induction for slc45a2 and tyr, respectively. At hatching this mutation frequency was also visible for both targeted genes, displaying a graded phenotype ranging from complete lack of pigmentation to partial loss and normal pigmentation. CRISPRslc45a2/Cas9 injected embryos showing a complete lack of pigmentation or just a few spots of pigments also lacked wild type sequences when assaying more than 80 (slc45a2) sequence clones from whole embryos. This indicates that CRISPR/Cas9 can induce double-allelic knockout in the F0 generation. However, types and frequency of indels might affect the phenotype. Therefore, the variation of indels was assayed in the graded pigmentation phenotypes produced by CRISPR/Cas9-slc45a2. The results show a tendency for fewer types of indels formed in juveniles completely lacking pigmentation compared to juveniles displaying partial pigmentation. Another interesting observation was a high degree of the same indel type in different juveniles. This study shows for the first time successful use of the CRISPR/Cas9 technology in a marine cold water species. Targeted double-allelic mutations were obtained and, though the level of mosaicism has to be considered, we demonstrate that F0 fish can be used for functional studies in Atlantic salmon.
了解关键蛋白质背后的生物学功能在大西洋鲑鱼中备受关注，这既是因为其具有很高的商业价值，也是由于其有趣的生活史。直到最近，鲑科鱼类的功能研究似乎都很困难。然而，最近利用CRISPR/Cas9（成簇规律间隔短回文重复序列/CRISPR相关蛋白）系统发现的靶向诱变技术，使得在很大程度上能够对大西洋鲑鱼进行功能研究。我们使用CRISPR/Cas9系统靶向两个参与色素沉着的基因，酪氨酸酶（tyr）和溶质载体家族45成员2（slc45a2）。在17体节阶段对胚胎进行突变率检测，所有注射胚胎中分别有40%和22%的胚胎显示出slc45a2和tyr的高度诱变。在孵化时，这两个靶向基因的突变频率也很明显，呈现出从完全无色素沉着到部分色素缺失和正常色素沉着的分级表型。当从整个胚胎中检测超过80个（slc45a2）序列克隆时，显示完全无色素沉着或仅有少数色素斑点的CRISPRslc45a2/Cas9注射胚胎也缺乏野生型序列。这表明CRISPR/Cas9可以在F0代诱导双等位基因敲除。然而，插入缺失的类型和频率可能会影响表型。因此，在CRISPR/Cas9-slc45a2产生的分级色素沉着表型中检测了插入缺失的变异。结果表明，与显示部分色素沉着的幼鱼相比，完全无色素沉着的幼鱼形成的插入缺失类型较少。另一个有趣的观察结果是，不同幼鱼中高度存在相同类型的插入缺失。这项研究首次展示了CRISPR/Cas9技术在海洋冷水物种中的成功应用。获得了靶向双等位基因突变，尽管必须考虑嵌合水平，但我们证明F0代鱼可用于大西洋鲑鱼的功能研究。
