@article {65297,
	title = {A versatile vector system for the fast generation of knock-in cell lines with CRISPR.},
	journal = {bioRxiv},
	year = {2020},
	month = {2020 Feb 06},
	abstract = {<p>Until recent advancements in genome editing via CRISPR/Cas9 technology, understanding protein function typically involved artificially overexpressing proteins of interest. Despite that CRISPR/Cas9 has ushered in a new era of possibilities for modifying endogenous genes with labeling tags (knock-in) to more accurately study proteins under physiological conditions, the technique is largely underutilized due to its tedious, multi-step process. Here we outline a homologous recombination system (FAST-HDR) to be used in combination with CRISPR/Cas9 that significantly simplifies and accelerates this process while introducing multiplexing to allow live-cell studies of 3 endogenous proteins within the same cell line. Furthermore, the recombination vectors are assembled in a single reaction that is enhanced for eliminating false positives and reduces the overall creation time for the knockin cell line from\&nbsp;~8 weeks to \&lt;15 days. Finally, the system utilizes a modular construction to allow for seamlessly swapping labeling tags to ensure flexibility according to the area under study. We validated this new methodology by developing advanced cell lines with 3 fluorescent-labeled endogenous proteins that support high-content phenotypic drug screening without using antibodies or exogenous staining. Therefore, Fast-HDR cell lines provide a robust alternative for studying multiple proteins of interest in live cells without artificially overexpressing labeled proteins.</p>
},
	doi = {10.1101/2020.02.06.927384},
	author = {Perez-Leal, Oscar and Nixon-Abell, Jonathon and Barrero, Carlos A. and Gordon, John and Rico, Mario C.}
}
