ＣＲＩＳＰＲ／Ｃａｓ９封入高分子集合体の構築と生体内デリバリーへの挑戦

采用基于荧光素酶的Cas9活性体系对不同阳离子聚合物的Cas9活性进行了评价。本实验由三种不同的质粒组成：sgrna表达质粒、含停止密码子的荧光素酶表达质粒和荧光素酶给体DNA质粒，由Moritoshi Sato教授（UTokyo）慷慨捐赠。将一种阳离子聚合物（PAsp(OCT/DET)）与Cas9信使RNA络合，然后将所得到的复合物转染人宫颈癌（HeLa）细胞。用荧光微孔板阅读器测定Cas9活性。与市售的转染剂脂质体3000相比，阳离子聚合物产生了相似的发光强度。这一结果表明，这种新型阳离子聚合物在体外转染Cas9 mRNA方面具有广阔的应用前景。

Cas9 activities of various cationic polymers were evaluated by luciferase-based Cas9 activity system. This assay consists of three different plasmids - sgRNA-expressing plasmid, Stop codon-contained luciferase-expressing plasmid, and luciferase donor DNA plasmid, which were kindly donated by Professor Moritoshi Sato (UTokyo). A cationic polymer (PAsp(OCT/DET)) was complexed with Cas9 messenger RNA and then the resulting polyplexes were transfected into human cervical cancer (HeLa) cells. The Cas9 activities were measured by a luminescence microplate reader. The cationic polymer elicited the similar luminescence intensity compared to commercially available transfection agent, lipofectamine 3000. This result indicates that the new cationic polymer is promising for in vitro Cas9 mRNA transfection.