@article {67813,
	title = {Multiplex Gene Tagging with CRISPR-Cas9 for Live-Cell Microscopy and Application to Study the Role of SARS-CoV-2 Proteins in Autophagy, Mitochondrial Dynamics, and Cell Growth.},
	journal = {The CRISPR Journal},
	year = {2021},
	month = {2021 Nov 30},
	abstract = {<p>The lack of efficient tools to label multiple endogenous targets in cell lines without staining or fixation has limited our ability to track physiological and pathological changes in cells over time via live-cell studies. Here, we outline the FAST-HDR vector system to be used in combination with CRISPR-Cas9 to allow visual live-cell studies of up to three endogenous proteins within the same cell line. Our approach utilizes a novel set of advanced donor plasmids for homology-directed repair and a streamlined workflow optimized for microscopy-based cell screening to create genetically modified cell lines that do not require staining or fixation to accommodate microscopy-based studies. We validated this new methodology by developing two advanced cell lines with three fluorescent-labeled endogenous proteins that support high-content imaging without using antibodies or exogenous staining. We applied this technology to study seven severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2/COVID-19) viral proteins to understand better their effects on autophagy, mitochondrial dynamics, and cell growth. Using these two cell lines, we were able to identify the protein ORF3a successfully as a potent inhibitor of autophagy, inducer of mitochondrial relocalization, and a growth inhibitor, which highlights the effectiveness of live-cell studies using this technology.</p>
},
	issn = {2573-1602},
	doi = {10.1089/crispr.2021.0041},
	author = {Perez-Leal, Oscar and Nixon-Abell, Jonathon and Barrero, Carlos A and Gordon, John C and Oesterling, James and Rico, Mario C}
}
@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.}
}
