Karp Hannah, Zoltek Madeline, Wasko Kevin, Vazquez Angel Luis, Brim Jinna, Ngo Wayne, Schepartz Alanna, Doudna Jennifer A
Department of Chemistry, University of California, Berkeley, CA 94720, United States.
Innovative Genomics Institute, University of California, Berkeley, CA 94720, United States.
Nucleic Acids Res. 2025 Feb 27;53(5). doi: 10.1093/nar/gkaf105.
Effective genome editing requires a sufficient dose of CRISPR-Cas9 ribonucleoproteins (RNPs) to enter the target cell while minimizing immune responses, off-target editing, and cytotoxicity. Clinical use of Cas9 RNPs currently entails electroporation into cells ex vivo, but no systematic comparison of this method to packaged RNP delivery has been made. Here we compared two delivery strategies, electroporation and enveloped delivery vehicles (EDVs), to investigate the Cas9 dosage requirements for genome editing. Using fluorescence correlation spectroscopy, we determined that >1300 Cas9 RNPs per nucleus are typically required for productive genome editing. EDV-mediated editing was >30-fold more efficient than electroporation, and editing occurs at least 2-fold faster for EDV delivery at comparable total Cas9 RNP doses. We hypothesize that differences in efficacy between these methods result in part from the increased duration of RNP nuclear residence resulting from EDV delivery. Our results directly compare RNP delivery strategies, showing that packaged delivery could dramatically reduce the amount of CRISPR-Cas9 RNPs required for experimental or clinical genome editing.
有效的基因组编辑需要足够剂量的CRISPR-Cas9核糖核蛋白（RNP）进入靶细胞，同时将免疫反应、脱靶编辑和细胞毒性降至最低。目前，Cas9 RNP的临床应用需要在体外将其电穿孔导入细胞，但尚未对该方法与包装的RNP递送进行系统比较。在此，我们比较了两种递送策略，即电穿孔和包膜递送载体（EDV），以研究基因组编辑所需的Cas9剂量要求。使用荧光相关光谱法，我们确定每个细胞核通常需要>1300个Cas9 RNP才能进行有效的基因组编辑。EDV介导的编辑效率比电穿孔高>30倍，并且在总Cas9 RNP剂量相当的情况下，EDV递送的编辑速度至少快2倍。我们推测，这些方法之间的效率差异部分源于EDV递送导致的RNP核内驻留时间延长。我们的结果直接比较了RNP递送策略，表明包装递送可以显著减少实验性或临床基因组编辑所需的CRISPR-Cas9 RNP的量。
