Optimization of Nuclear Localization Signal Composition Improves CRISPR-Cas12a Editing Rates in Human Primary Cells.
Optimization of Nuclear Localization Signal Composition Improves CRISPR-Cas12a Editing Rates in Human Primary Cells.
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DOI：
10.1089/genbio.2022.0003
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发表时间：
2022-06
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通讯作者：
Kevin Luk;Pengpeng Liu;Jing Zeng;Yetao Wang;Stacy A. Maitland;Feston Idrizi;Karthikeyan Ponnienselvan;L. Zhu;J. Luban;D. E. Bauer;S. Wolfe
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作者：
Kevin Luk;Pengpeng Liu;Jing Zeng;Yetao Wang;Stacy A. Maitland;Feston Idrizi;Karthikeyan Ponnienselvan;L. Zhu;J. Luban;D. E. Bauer;S. Wolfe
V型CRISPR-Cas 12 a系统是用于特定基因组编辑应用的有吸引力的Cas9替代核酸酶平台。然而，先前的研究表明，在原代细胞中Cas 12 a和Cas9之间的总体活性存在差距。1在此，我们描述了对Cas 12 a的NLS组成和结构的优化，以促进在人转化细胞系（HEK 293 T，Jurkat和K562细胞）和原代细胞（NK细胞和CD 34 + HSPC）中的高效靶向诱变，而不考虑Cas 12 a直系同源物。我们的3xNLS Cas 12 a架构产生了最强大的编辑平台。Cas 12 a在NK细胞和CD 34 + HSPC中的编辑活性改善导致与靶基因编辑相关的显著表型变化。最后，我们证明了Cas 12 a的NLS组成和结构的优化不会增加HEK 293 T或CD 34 + HSPC中潜在脱靶位点的编辑。我们的新Cas 12 a NLS变体为治疗性基因组编辑提供了改进的核酸酶平台。
Type V CRISPR-Cas12a systems are an attractive Cas9-alternative nuclease platform for specific genome editing applications. However, previous studies demonstrate that there is a gap in overall activity between Cas12a and Cas9 in primary cells.1 Here we describe optimization to the NLS composition and architecture of Cas12a to facilitate highly efficient targeted mutagenesis in human transformed cell lines (HEK293T, Jurkat, and K562 cells) and primary cells (NK cells and CD34+ HSPCs), regardless of Cas12a ortholog. Our 3xNLS Cas12a architecture resulted in the most robust editing platform. The improved editing activity of Cas12a in both NK cells and CD34+ HSPCs resulted in pronounced phenotypic changes associated with target gene editing. Lastly, we demonstrated that optimization of the NLS composition and architecture of Cas12a did not increase editing at potential off-target sites in HEK293T or CD34+ HSPCs. Our new Cas12a NLS variant provides an improved nuclease platform for therapeutic genome editing.