Luminescent peptide tagging enables efficient screening for CRISPR-mediated knock-in in human induced pluripotent stem cells.

ABSTRACT: Human pluripotent stem cells are increasingly used for CRISPR-mediated gene targeting in efforts to generate models of human diseases. This is a challenging task because of the high sensitivity of these cells to suboptimal conditions, including CRISPR-associated DNA damage and subsequent rounds of single-cell cloning. We sought to develop a sensitive method that enables rapid screening of CRISPR targeted cells, while preserving cell viability and eliminating the need for expensive sequencing of a large number of clones. A protocol was designed in which the luminescent peptide tag, HiBiT, is appended to the extracellular portion of an inert surface membrane protein (CD46), using synthetic CRISPR reagents and a widely distributed human induced pluripotent stem cell (iPSC) line. We find that

SUBMITTER: Madsen RR 

PROVIDER: S-EPMC6640264 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature