以益生大腸桿菌衍生之奈米載體作爲CRISPR-Cas12b之遞送系統

Abstract

基因編輯系統CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins)自其發現以來，在醫療領域上具有重大應用與發展潛力，特別是在基因治療。然而CRISPR-Cas需要一個安全可靠、可廣泛、製造經濟化的運送系統，方以有效用於生物體的基因編輯與治療，目前的運送載體，例如病毒載體或人造聚合物往往面臨諸多挑戰，如安全因素或合成上的繁瑣複雜等。除此之外，CRISPR-Cas本身的遞送形式也是一個考量重點，爲了避免外源基因插入宿主基因體的風險，使用信使核糖核酸 (mRNA) 或核糖蛋白 (ribonucleoprotein, RNP) 是較爲安全的形式。本研究意在建立一種新型的益生菌之CRISPR-Cas RNP遞送系統，以益生菌衍生之奈米載體作爲包裹和傳遞RNP的載體。具體來說，益生菌Escherichia coli Nissle 1917 (EcN) 以基因敲落的方式，令其在分裂時產生不再分裂的微小細胞 (minicells)，同時讓EcN表達外源蛋白Cas12b使其在胞內組裝Cas12b RNP。這些微小細胞會藉由表達外膜蛋白invasin以結合哺乳類細胞表面，並藉由表現listeriolysin O來釋放細菌表達的蛋白進入細胞內部，一旦微小細胞被吞入並在細胞的胞內體中裂解，Cas12b RNP便可藉由核定位序列 (nucleus localization signal, NLS) 的幫助，運送到細胞核內完成基因編輯。同時在研究中，我們建構了CRISPR編輯報道細胞株 DGG.HeLa，以功能增益 (grain-of-function) 的方式來確認細胞是否有被成功編輯，在基因被成功編輯後，細胞才會生產綠色螢光蛋白 (green fluorescence protein; GFP) 。更具結果顯示，經由 minicells 運輸 Cas12b-sgRNA，我們能成功編輯 DGG.HeLa，並產生最高約5% 之永久綠色系細胞株。在未來我們將繼續調整 minicells 系統，增加編輯效率與生醫應用性。

CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) systems have an emerging utilization and research since its discovery, especially in gene therapy. A safe, universal, and affordable delivery platform is required when encountering in vivo editing. Current platforms such as viral vector and artificial polymer face several challenges, such as safety issues and complication in production. In addition, the form of delivered CRISPR-Cas is an issue to concern. It is commonly accepted that delivering RNP (ribonucleoprotein) and mRNA of CRIPSR-Cas are safer than delivering CRISPR-Cas DNA, to avoid possible gene integration. Here, we utilize a bioengineered probiotic-derived nanocarrier to deliver functional CRISPR-Cas12b RNP. Specifically, Escherichia coli Nissle 1917 (EcN) is engineered to produce non-proliferating minicells by gene knockout, as well as to express functional Cas12b RNP within the minicells. These minicells are capable of binding mammalian cells by expressing Yersinia outer membrane protein invasin, and are able to escape from endosome by expressing listeriolysin O. Once the minicells are engulfed and lysed within the targeting cell, Cas12b RNP would be transferred into nucleus by the assist of nuclear localization protein (NLS) to conduct further genome editing. In this study, we constructed a CRISPR-reporting cell line DGG.HeLa to specifically producing green fluorescence protein (GFP) only after CRISPR-editing via Cas12b-sgRNA minicells. As the result demonstrated in the research, the GFP-expressing stable DGG.HeLa cell line was created at the success rate of 5% via the engineered minicell system. We anticipate that further development of the CRISPR-minicell delivery system will increase the editing efficiency of future biomedical applications.