Shui Sailan, Wang Shaojie, Liu Jia
Shanghai Institute for Advanced Immunochemical Studies, School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
Bioengineering (Basel). 2022 Feb 21;9(2):83. doi: 10.3390/bioengineering9020083.
The emergence of CRISPR-Cas9 technology has revolutionized both basic and translational biomedical research. For Cas9 nuclease to exert genome editing activity, nuclear localization signal (NLS) derived from simian virus 40 (SV40) T antigen is commonly installed as genetic fusion to direct the intracellular Cas9 proteins to the nucleus of cells. Notably, previous studies have shown that multiple SV40 NLS fusion can improve the targeting activity of Cas9-derived genome-editing and base-editing tools. In addition, the multi-NLS fusion can increase the intracellular activity of Cas9 in the forms of both constitutive expression and directly delivered Cas9-guide RNA ribonucleoprotein (RNP) complex. However, the relationship between NLS fusion and intracellular Cas9 activity has not been fully understood, including the dependency of activity on the number or organization of NLS fusion. In the present study, we constructed and purified a set of Streptococcus pyogenes Cas9 (SpCas9) variants containing one to four NLS repeats at the N- or C-terminus of the proteins and systematically analyzed the effects of multi-NLS fusion on the activity of SpCas9 RNPs. It was found that multi-NLS fusion could improve the intracellular activity as lipofected or nucleofected Cas9 RNPs. Importantly, multi-NLS fusion could enhance the genome-editing activity of SpCas9 RNPs in primary and stem/progenitor cells and mouse embryos.
CRISPR-Cas9技术的出现彻底改变了基础和转化生物医学研究。为了使Cas9核酸酶发挥基因组编辑活性，通常会将源自猴病毒40（SV40）T抗原的核定位信号（NLS）作为基因融合物安装，以将细胞内的Cas9蛋白引导至细胞核。值得注意的是，先前的研究表明，多个SV40 NLS融合可以提高Cas9衍生的基因组编辑和碱基编辑工具的靶向活性。此外，多NLS融合可以以组成型表达和直接递送的Cas9引导RNA核糖核蛋白（RNP）复合物的形式增加Cas9的细胞内活性。然而，NLS融合与细胞内Cas9活性之间的关系尚未完全理解，包括活性对NLS融合数量或组织的依赖性。在本研究中，我们构建并纯化了一组化脓性链球菌Cas9（SpCas9）变体，这些变体在蛋白质的N端或C端含有一到四个NLS重复序列，并系统地分析了多NLS融合对SpCas9 RNPs活性的影响。结果发现，多NLS融合可以提高作为脂质转染或核转染的Cas9 RNPs的细胞内活性。重要的是，多NLS融合可以增强SpCas9 RNPs在原代细胞、干细胞/祖细胞和小鼠胚胎中的基因组编辑活性。
