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.
CRISPR J
; 4(6): 854-871, 2021 12.
Article
in English
| MEDLINE | ID: covidwho-1545880
ABSTRACT
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.
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Autophagy
/
Gene Targeting
/
Mitochondrial Dynamics
/
CRISPR-Cas Systems
/
Viroporin Proteins
/
SARS-CoV-2
/
COVID-19
Type of study:
Prognostic study
Limits:
Humans
Language:
English
Journal:
CRISPR J
Year:
2021
Document Type:
Article
Affiliation country:
Crispr.2021.0041
Similar
MEDLINE
...
LILACS
LIS