Genetic Knock-Ins of Endogenous Fluorescent Tags in RAW 264.7 Murine Macrophages Using CRISPR/Cas9 Genome Editing
CRISPR/Cas9 genome editing is a widely used tool for creating genetic knock-ins, which allow for endogenous tagging of genes. This is in contrast with random insertion using viral vectors, where expression of the inserted transgene changes the total copy number of a gene in a cell and does not refle...
| Main Authors: | , , |
|---|---|
| 格式: | Article |
| 語言: | English |
| 出版: |
Bio-protocol LLC
2024-03-01
|
| 叢編: | Bio-Protocol |
| 在線閱讀: | https://bio-protocol.org/en/bpdetail?id=4960&type=0 |
| _version_ | 1831869501023453184 |
|---|---|
| author | Beverly Naigles Jan Soroczynski Nan Hao |
| author_facet | Beverly Naigles Jan Soroczynski Nan Hao |
| author_sort | Beverly Naigles |
| collection | DOAJ |
| description | CRISPR/Cas9 genome editing is a widely used tool for creating genetic knock-ins, which allow for endogenous tagging of genes. This is in contrast with random insertion using viral vectors, where expression of the inserted transgene changes the total copy number of a gene in a cell and does not reflect the endogenous chromatin environment or any trans-acting regulation experienced at a locus. There are very few protocols for endogenous fluorescent tagging in macrophages. Here, we describe a protocol to design and test CRISPR guide RNAs and donor plasmids, to transfect them into RAW 264.7 mouse macrophage-like cells using the Neon transfection system and to grow up clonal populations of cells containing the endogenous knock-in at various loci. We have used this protocol to create endogenous fluorescent knock-ins in at least six loci, including both endogenously tagging genes and inserting transgenes in the Rosa26 and Tigre safe harbor loci. This protocol uses circular plasmid DNA as the donor template and delivers the sgRNA and Cas9 as an all-in-one expression plasmid. We designed this protocol for fluorescent protein knock-ins; it is best used when positive clones can be identified by fluorescence. However, it may be possible to adapt the protocol for non-fluorescent knock-ins. This protocol allows for the fairly straightforward creation of clonal populations of macrophages with tags at the endogenous loci of genes. We also describe how to set up imaging experiments in 24-well plates to track fluorescence in the edited cells over time.Key features• CRISPR knock-in of fluorescent proteins in RAW 264.7 mouse macrophages at diverse genomic loci.• This protocol is optimized for the use of the Neon transfection system.• Includes instructions for growing up edited clonal populations from single cells with one single-cell sorting step and efficient growth in conditioned media after cell sorting.• Designed for knocking in fluorescent proteins and screening transfected cells byFACS, but modification for non-fluorescent knock-ins may be possible.Graphical overview |
| first_indexed | 2025-03-21T19:11:21Z |
| format | Article |
| id | doaj.art-72649e21b51a42999df9f3d255afd9b6 |
| institution | Directory Open Access Journal |
| issn | 2331-8325 |
| language | English |
| last_indexed | 2025-03-21T19:11:21Z |
| publishDate | 2024-03-01 |
| publisher | Bio-protocol LLC |
| record_format | Article |
| series | Bio-Protocol |
| spelling | doaj.art-72649e21b51a42999df9f3d255afd9b62024-06-04T09:54:50ZengBio-protocol LLCBio-Protocol2331-83252024-03-0114610.21769/BioProtoc.4960Genetic Knock-Ins of Endogenous Fluorescent Tags in RAW 264.7 Murine Macrophages Using CRISPR/Cas9 Genome EditingBeverly Naigles0Jan Soroczynski1Nan Hao2Deparment of Molecular Biology, University of California San Diego, La Jolla, CA, USALaboratory of Genome Architecture and Dynamics, The Rockefeller University, New York, NY, USADeparment of Molecular Biology, University of California San Diego, La Jolla, CA, USADepartment of Bioengineering, University of California San Diego, La Jolla, CA, USACRISPR/Cas9 genome editing is a widely used tool for creating genetic knock-ins, which allow for endogenous tagging of genes. This is in contrast with random insertion using viral vectors, where expression of the inserted transgene changes the total copy number of a gene in a cell and does not reflect the endogenous chromatin environment or any trans-acting regulation experienced at a locus. There are very few protocols for endogenous fluorescent tagging in macrophages. Here, we describe a protocol to design and test CRISPR guide RNAs and donor plasmids, to transfect them into RAW 264.7 mouse macrophage-like cells using the Neon transfection system and to grow up clonal populations of cells containing the endogenous knock-in at various loci. We have used this protocol to create endogenous fluorescent knock-ins in at least six loci, including both endogenously tagging genes and inserting transgenes in the Rosa26 and Tigre safe harbor loci. This protocol uses circular plasmid DNA as the donor template and delivers the sgRNA and Cas9 as an all-in-one expression plasmid. We designed this protocol for fluorescent protein knock-ins; it is best used when positive clones can be identified by fluorescence. However, it may be possible to adapt the protocol for non-fluorescent knock-ins. This protocol allows for the fairly straightforward creation of clonal populations of macrophages with tags at the endogenous loci of genes. We also describe how to set up imaging experiments in 24-well plates to track fluorescence in the edited cells over time.Key features• CRISPR knock-in of fluorescent proteins in RAW 264.7 mouse macrophages at diverse genomic loci.• This protocol is optimized for the use of the Neon transfection system.• Includes instructions for growing up edited clonal populations from single cells with one single-cell sorting step and efficient growth in conditioned media after cell sorting.• Designed for knocking in fluorescent proteins and screening transfected cells byFACS, but modification for non-fluorescent knock-ins may be possible.Graphical overviewhttps://bio-protocol.org/en/bpdetail?id=4960&type=0 |
| spellingShingle | Beverly Naigles Jan Soroczynski Nan Hao Genetic Knock-Ins of Endogenous Fluorescent Tags in RAW 264.7 Murine Macrophages Using CRISPR/Cas9 Genome Editing Bio-Protocol |
| title | Genetic Knock-Ins of Endogenous Fluorescent Tags in RAW 264.7 Murine Macrophages Using CRISPR/Cas9 Genome Editing |
| title_full | Genetic Knock-Ins of Endogenous Fluorescent Tags in RAW 264.7 Murine Macrophages Using CRISPR/Cas9 Genome Editing |
| title_fullStr | Genetic Knock-Ins of Endogenous Fluorescent Tags in RAW 264.7 Murine Macrophages Using CRISPR/Cas9 Genome Editing |
| title_full_unstemmed | Genetic Knock-Ins of Endogenous Fluorescent Tags in RAW 264.7 Murine Macrophages Using CRISPR/Cas9 Genome Editing |
| title_short | Genetic Knock-Ins of Endogenous Fluorescent Tags in RAW 264.7 Murine Macrophages Using CRISPR/Cas9 Genome Editing |
| title_sort | genetic knock ins of endogenous fluorescent tags in raw 264 7 murine macrophages using crispr cas9 genome editing |
| url | https://bio-protocol.org/en/bpdetail?id=4960&type=0 |
| work_keys_str_mv | AT beverlynaigles geneticknockinsofendogenousfluorescenttagsinraw2647murinemacrophagesusingcrisprcas9genomeediting AT jansoroczynski geneticknockinsofendogenousfluorescenttagsinraw2647murinemacrophagesusingcrisprcas9genomeediting AT nanhao geneticknockinsofendogenousfluorescenttagsinraw2647murinemacrophagesusingcrisprcas9genomeediting |