A Small-Molecule Mask for Traceless Protein Delivery

项目总结/摘要 CRISPR/Cas9系统已迅速成为迄今为止发现的最强大的基因编辑工具。许多 采用该系统的早期论文已经利用了编码Cas9蛋白和指导RNA的质粒 （sgRNA）。这些质粒通常在病毒外壳中递送，对于整个质粒来说，病毒外壳通常太小。 质粒，需要多个片段分开运输。质粒掺入的另一个缺点是 是蛋白质和RNA的持续表达，这通常导致不期望的脱靶效应。一 有希望的替代方案是直接递送Cas9·sgRNA复合物。各种各样的方法已经被 开发用于递送这种复合物，包括脂质纳米颗粒和细胞穿透肽，但所有 迄今为止的方法导致低效的基因编辑或不适用于体内（或两者）。我们实验室最近的工作 证明了苯并氧杂硼杂环戊烯官能团的独特化学性质使其能够递送蛋白质 有效地直接进入胞质溶胶。将这种独特的化学性质与重氮基序的反应性相结合， 使我们能够开发出可以通过酯化反应掩蔽Cas9羧酸酯的小分子。一系列 这些递送载体将被合成并与Cas9·sgRNA复合物反应以评估它们的效果 复合物稳定性和蛋白质-RNA结合。最初的实验将集中在敲除GFP基因， 产生GFP的HEK细胞。一旦确定了最佳运载工具和条件， 载体将在未来的乳腺癌转移研究中用作工具。胶原脯氨酰4-羟化酶 已经证明在转移过程中是重要的，我们预计敲除这个基因， 通过CRISPR/Cas9系统将基因导入将是一种有效的方法。最后，我们的交付战略可以提供 这是一种简单的方法，不仅用于递送Cas9·sgRNA复合物，而且用于递送各种蛋白质。

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.