CRISPR/Cas9-mediated LRP10 Knockout in HuTu-80 and HEK 293T Cell Lines

Abstract

Loss-of-function (LoF) variants in the low-density lipoprotein receptor-related protein 10 gene ( LRP10 ) have been recently implicated in the development of neurodegenerative diseases, including Parkinson's disease (PD), PD dementia (PDD), and dementia with Lewy bodies (DLB). However, despite the genetic evidence, little is known about the LRP10 protein function in health and disease. Here, we describe a detailed protocol to efficiently generate a LRP10 LoF model in two independent LRP10-expressing cell lines, HuTu-80 and HEK 293T, using the CRISPR/Cas9 genome-editing tool. Our method efficiently generates bi-allelic LRP10 knockout (KO), which can be further utilized to elucidate the physiological LRP10 protein function and to model some aspects of neurodegenerative disorders. Graphical abstract: CRISPR/Cas9 workflow for the generation of the LRP10 KO. (1) Designed single guide RNA (sgRNA) is cloned into CRISPR/Cas9 px458 plasmid. (2) Cells are transfected with the CRISPR/Cas9 plasmid containing sgRNA. (3) Two days post transfection, cells are dissociated and sorted as single cells by fluorescence-activated cell sorting (FACS). (4) After several weeks, expanded clonal lines are (5) verified with Sanger sequencing for the presence of INDELs ( in sertions or del etions), RT-qPCR for the amounts of LRP10 mRNA transcript, and Western blotting for the analysis of the LRP10 protein levels.

Keywords: CRISPR/Cas9; Gene editing; HEK 293T; HuTu-80; Knockout; LRP10.