Eugene Bell Center, Marine Biological Laboratory, Woods Hole, MA 02543, USA.
Department of Neurosurgery and Stanford Neurosciences Institute, Stanford University, Stanford, CA 94305, USA.
Curr Biol. 2023 Jul 10;33(13):2774-2783.e5. doi: 10.1016/j.cub.2023.05.066. Epub 2023 Jun 20.
Cephalopods are remarkable among invertebrates for their cognitive abilities, adaptive camouflage, novel structures, and propensity for recoding proteins through RNA editing. Due to the lack of genetically tractable cephalopod models, however, the mechanisms underlying these innovations are poorly understood. Genome editing tools such as CRISPR-Cas9 allow targeted mutations in diverse species to better link genes and function. One emerging cephalopod model, Euprymna berryi, produces large numbers of embryos that can be easily cultured throughout their life cycle and has a sequenced genome. As proof of principle, we used CRISPR-Cas9 in E. berryi to target the gene for tryptophan 2,3 dioxygenase (TDO), an enzyme required for the formation of ommochromes, the pigments present in the eyes and chromatophores of cephalopods. CRISPR-Cas9 ribonucleoproteins targeting tdo were injected into early embryos and then cultured to adulthood. Unexpectedly, the injected specimens were pigmented, despite verification of indels at the targeted sites by sequencing in injected animals (G0s). A homozygote knockout line for TDO, bred through multiple generations, was also pigmented. Surprisingly, a gene encoding indoleamine 2,3, dioxygenase (IDO), an enzyme that catalyzes the same reaction as TDO in vertebrates, was also present in E. berryi. Double knockouts of both tdo and ido with CRISPR-Cas9 produced an albino phenotype. We demonstrate the utility of these albinos for in vivo imaging of Ca signaling in the brain using two-photon microscopy. These data show the feasibility of making gene knockout cephalopod lines that can be used for live imaging of neural activity in these behaviorally sophisticated organisms.
头足类动物在无脊椎动物中以其认知能力、适应性伪装、新颖的结构和通过 RNA 编辑重新编码蛋白质的倾向而引人注目。然而，由于缺乏遗传上可操作的头足类模型，这些创新的机制仍知之甚少。基因组编辑工具如 CRISPR-Cas9 允许在不同物种中进行靶向突变，以更好地将基因与功能联系起来。一种新兴的头足类模型，Euprymna berryi，产生大量胚胎，可以在整个生命周期中轻松培养，并且具有测序的基因组。作为原理的证明，我们使用 CRISPR-Cas9 在 E. berryi 中靶向色氨酸 2,3 加双氧酶（TDO）基因，该基因是形成章鱼胺的必需酶，章鱼胺是头足类动物眼睛和色素细胞中存在的色素。针对 tdo 的 CRISPR-Cas9 核糖核蛋白被注射到早期胚胎中，然后培养至成年。出乎意料的是，尽管在注射动物（G0s）中通过测序验证了靶位点的插入缺失，但注射标本仍有色素沉着。通过多代繁殖培育的 TDO 纯合敲除系也有色素沉着。令人惊讶的是，E. berryi 中还存在编码吲哚胺 2,3,双氧酶（IDO）的基因，该酶在脊椎动物中催化与 TDO 相同的反应。使用 CRISPR-Cas9 对 tdo 和 ido 进行双重敲除产生了白化表型。我们使用双光子显微镜证明了这些白化体在大脑中的 Ca 信号体内成像的实用性。这些数据表明，制造可用于这些行为复杂生物的神经活动活体成像的基因敲除头足类动物系是可行的。
