Cell, Molecular biology and Genomics Group, Department of Biology, Norwegian University of Science and Technology, N-7491, Trondheim, Norway.
Sci Rep. 2018 Sep 26;8(1):14401. doi: 10.1038/s41598-018-32342-0.
The CRISPR/Cas9 technology has opened the possibility for targeted genome editing in various organisms including diatom model organisms. One standard method for delivery of vectors to diatom cells is by biolistic particle bombardment. Recently delivery by conjugation was added to the tool-box. An important difference between these methods is that biolistic transformation results in transgene integration of vector DNA into the algae genome, whereas conjugative transformation allows the vector to be maintained as an episome in the recipient cells. In this study, we have used both transformation methods to deliver the CRISPR/Cas9 system to the marine diatom Phaeodactylum tricornutum aiming to induce mutations in a common target gene. This allowed us to compare the two CRISPR/Cas9 delivery systems with regard to mutation efficiency, and to assess potential problems connected to constitutive expression of Cas9. We found that the percentage of CRISPR-induced targeted biallelic mutations are similar for both methods, but an extended growth period might be needed to induce biallelic mutations when the CRISPR/Cas9 system is episomal. Independent of the CRISPR/Cas9 vector system, constitutive expression of Cas9 can cause re-editing of mutant lines with small indels. Complications associated with the biolistic transformation system like the permanent and random integration of foreign DNA into the host genome and unstable mutant lines caused by constitutive expression of Cas9 can be avoided using the episomal CRISPR/Cas9 system. The episomal vector can be eliminated from the diatom cells by removal of selection pressure, resulting in transient Cas9 expression and non-transgenic mutant lines. Depending on legislation, such lines might be considered as non-GMOs.
CRISPR/Cas9 技术为包括硅藻模式生物在内的各种生物的靶向基因组编辑开辟了可能性。将载体递送至硅藻细胞的一种标准方法是通过生物弹道粒子轰击。最近,共轭传递被添加到工具盒中。这两种方法的一个重要区别是,生物弹道转化导致载体 DNA 整合到藻类基因组中,而共轭转化允许载体作为染色体外体在受体细胞中维持。在这项研究中,我们使用了这两种转化方法将 CRISPR/Cas9 系统递送至海洋硅藻三角褐指藻,旨在诱导常见靶基因的突变。这使我们能够比较两种 CRISPR/Cas9 传递系统在突变效率方面的差异,并评估与 Cas9 组成型表达相关的潜在问题。我们发现,两种方法诱导 CRISPR 诱导的靶向双等位基因突变的百分比相似,但当 CRISPR/Cas9 系统为染色体外体时,可能需要延长生长周期才能诱导双等位基因突变。无论 CRISPR/Cas9 载体系统如何,Cas9 的组成型表达都可能导致带有小插入缺失的突变体的重新编辑。生物弹道转化系统相关的并发症,如外源 DNA 永久性和随机整合到宿主基因组中以及 Cas9 的组成型表达导致不稳定的突变体系,可以通过使用染色体外体 CRISPR/Cas9 系统来避免。可以通过去除选择压力从硅藻细胞中消除染色体外体载体,从而导致瞬时 Cas9 表达和非转基因突变体系。根据法规,此类系可能被视为非转基因生物。