Luminescent peptide tagging enables efficient screening for CRISPR-mediated knock-in in human induced pluripotent stem cells.
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 this approach substantially reduces labour-intensive screening of CRISPR-targeted iPSCs and minimises the number of subcloning steps. Successfully edited iPSCs could be identified within a week of targeting, based only on extracellular luminescence detection in live cells. The total screening time in each round was less than 30 minutes and no sequencing was required. This method can be developed further to serve as a highly sensitive co-selection strategy in CRISPR knock-in experiments, particularly in the context of challenging cell lines.
['Madsen|Ralitsa R|RR|https://orcid.org/0000-0001-8844-5167', 'Semple|Robert K|RK|https://orcid.org/0000-0001-6539-3069']
tagging screening knock-in human pluripotent stem cells CRISPR HiBiT iPSCs