磁性肽印迹壳聚糖复合纳米颗粒对抗原的识别用于从转染细胞中提取 CRISPR/dCas9a 蛋白。

Epitope recognition of magnetic peptide-imprinted chitosan composite nanoparticles for the extraction of CRISPR/dCas9a proteins from transfected cells.

机构信息

Department of Materials Science and Engineering, I-Shou University, Kaohsiung 84001, Taiwan.

Division of Pulmonary Medicine, Department of Internal Medicine, Armed-Forces Zuoying General Hospital, Kaohsiung 81342, Taiwan.

出版信息

Nanotechnology. 2021 Apr 30;32(18):18LT02. doi: 10.1088/1361-6528/abde00.

DOI:10.1088/1361-6528/abde00

PMID:33472179

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9) technology is a powerful method for genetic modification (and regulation) that is of great current interest. The development of new, economical methods of detecting and extracting Cas9 (and/or dCas9) from transfected cells is thus an important advance. In this work, we employed molecular imprinting, using two peptides from the Cas9 protein, to make magnetic peptide-imprinted chitosan nanoparticles. dCas9 was encoded in a plasmid which was then transfected into human embryonic kidney (HEK293T) cells. The expression of dCas9 protein was measured by using total protein kits. Finally, the imprinted nanoparticles were used to extract dCas9 from transfected cell homogenates.

摘要

簇状规律间隔短回文重复（CRISPR）/CRISPR 相关蛋白（Cas9）技术是一种强大的遗传修饰（和调控）方法，目前受到广泛关注。因此，开发新的、经济的方法来检测和从转染细胞中提取 Cas9（和/或 dCas9）是一项重要的进展。在这项工作中，我们使用 Cas9 蛋白的两个肽段进行分子印迹，制备了磁性肽印迹壳聚糖纳米粒子。dCas9 被编码在质粒中，然后转染到人胚肾（HEK293T）细胞中。使用总蛋白试剂盒测量 dCas9 蛋白的表达。最后，印迹纳米粒子被用于从转染细胞匀浆中提取 dCas9。