Department of Integrative Marine Ecology, Stazione Zoologica Anton Dohrn, Naples, Italy.
Institute of Biosciences and BioResources, CNR, Naples, Italy.
Methods Mol Biol. 2022;2498:327-336. doi: 10.1007/978-1-0716-2313-8_18.
The CRISPR/Cas9 system coupled with proteolistics is a DNA-free nuclear transformation method based on the introduction of ribonucleoprotein (RNP) complexes into cells. The method has been set up for diatoms as an alternative to genetic transformation via biolistics and has the advantages of reducing off-target mutations, limiting the working time of the Cas9 endonuclease, and overcoming the occurrence of random insertions of the transgene in the genome. We present a point-by-point description of the protocol with modifications that make it more cost-effective, by reducing the amount of the enzyme while maintaining a comparable efficiency to the original protocol, and with an increased concentration of the selective drug which allows to reduce false positives.
CRISPR/Cas9 系统与蛋白质组学相结合，是一种基于将核糖核蛋白 (RNP) 复合物导入细胞的无 DNA 核转化方法。该方法已为硅藻建立，作为生物弹粒转化的替代方法，具有减少脱靶突变、限制 Cas9 内切酶的工作时间以及克服转基因在基因组中随机插入的优点。我们介绍了该方案的详细描述，并进行了修改，通过减少酶的用量，同时保持与原始方案相当的效率，以及增加选择药物的浓度，从而降低假阳性率，使其更具成本效益。
