Apr 18, 2022
Generation of knockout and rescue cell lines using CRISPR-Cas9 genome editing
- William Hancock-Cerutti1,2,3,4,5,
- Jun Hyun Park1,2,3,6,5,
- Pietro De Camilli1,2,3,5
- 1Departments of Neuroscience and of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 2Howard Hughes Medical Institute;
- 3Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 4Interdisciplinary Neuroscience Program and MD-PhD Program, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 5Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815;
- 6Interdisciplinary Neuroscience Program, Yale University School of Medicine, New Haven, Connecticut 06510, USA
External link: https://doi.org/10.1083/jcb.202106046
Protocol Citation: William Hancock-Cerutti, Jun Hyun Park, Pietro De Camilli 2022. Generation of knockout and rescue cell lines using CRISPR-Cas9 genome editing. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lynx5wvx9/v1
Manuscript citation:
William Hancock-Cerutti, Zheng Wu, Peng Xu, Narayana Yadavalli, Marianna Leonzino, Arun Kumar Tharkeshwar, Shawn M. Ferguson, Gerald S. Shadel, Pietro De Camilli (2022) ER-lysosome lipid transfer protein VPS13C/PARK23 prevents aberrant mtDNA-dependent STING signaling.Journal of Cell Biology doi: 10.1083/jcb.202106046
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: April 18, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 60895
Keywords: ASAPCRN, cas9 genome editing this protocol, cas9 genome editing, insertions by crispr, using crispr, crispr, genetic modification of cultured cell, genetic modification, rescue cell line, mediated repair, genomic dna, cultured cell, cas9, mutation, dna, repair of single base deletion, transfection, cell, screening of single clone, generation of knockout
Abstract
This protocol describes the genetic modification of cultured cells using CRISPR-Cas9, including synthesis of reagents, transfection, selection and screening of single clones, and sequencing of genomic DNA to confirm mutations. In addition, this protocol describes CRISPR-Cas9 mediated repair of single base deletions or insertions by CRISPR-Cas9.
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