A Small-Molecule Mask for Traceless Protein Delivery

项目摘要/摘要 CRISPR/Cas9系统已迅速成为迄今发现的最强大的基因编辑工具。许多 早期使用该系统的论文利用了编码Cas9蛋白和引导RNA的质粒 (SgRNA)。这些质粒通常以病毒壳的形式传递，这种壳往往太小，不能完全 质粒，需要多个片段分开运输。质粒掺入的另一个缺点 是蛋白质和RNA的持续表达，这往往会导致不希望看到的脱靶效应。一个 有希望的替代方案是直接交付Cas9·sgRNA复合体。已经有了各种各样的方法 为传递这种复合体而开发的，包括脂质纳米粒和细胞穿透肽，但所有 到目前为止，这些方法导致基因编辑效率低下或不适用于体内(或两者兼而有之)。我们实验室最近的工作是 证明了苯并氧硼环官能团的独特化学特性使其能够输送蛋白质 高效地直接进入细胞质。将这种独特的化学反应与重氮基序的反应性结合起来将 允许我们开发能够通过酯化反应掩盖Cas9羧酸盐的小分子。一系列 这些递送载体将被合成并与Cas9·sgRNA复合体反应，以评估其效果 关于络合物的稳定性和蛋白质与RNA的结合。最初的实验将集中在敲除GFP基因。 产生绿色荧光蛋白的HEK细胞。一旦确定了最佳的输送工具和条件， Vehicle将被用作未来乳腺癌转移研究的工具。胶原蛋白Pro-4-羟基酶 已经被证明在转移过程中具有重要意义，我们预计这种敲除 通过CRISPR/Cas9系统进行基因检测将是一种有效的方法。最后，我们的交付策略可以提供 简单的方法不仅可以提供Cas9·sgRNA复合体，还可以提供各种蛋白质。

PROJECT SUMMARY/ABSTRACT The CRISPR/Cas9 system has quickly emerged as the most robust gene editing tool yet discovered. Many early papers employing this system have utilized a plasmid that encodes the Cas9 protein and the guide RNA (sgRNA). These plasmids are typically delivered in viral shells that can often be too small for the entire plasmid, requiring multiple segments to be transported separately. Another drawback of plasmid incorporation is the continual expression of the protein and RNA, which often leads to undesired off-target effects. A promising alternative is the delivery of the Cas9·sgRNA complex directly. A variety of methods have been developed for the delivery of this complex, including lipid nanoparticles and cell-penetrating peptides, but all methods so far result in inefficient gene editing or are inapplicable in vivo (or both). Recent work in our lab has demonstrated that the unique chemistry of the benzoxaborole functional group allows it to deliver proteins efficiently and directly into the cytosol. Combining this unique chemistry with the reactivity of a diazo motif will allow us to develop small molecules that can mask Cas9 carboxylates via an esterification reaction. A series of these delivery vehicles will be synthesized and reacted with the Cas9·sgRNA complex to evaluate their effect on complex stability and protein–RNA binding. Initial experiments will focus on knocking out the GFP gene in GFP-producing HEK cells. Once the optimal delivery vehicle and conditions have been determined, that vehicle will be employed as a tool in future studies of breast cancer metastasis. Collagen prolyl 4-hydroxylase has been demonstrated to be significant in the metastasis process, and we anticipate that knock-out of this gene via a CRISPR/Cas9 system would be an effective approach. Finally, our delivery strategy could provide a straightforward method for delivering not only Cas9·sgRNA complexes, but also a wide variety of proteins.