reconstitution of functional entities and therefore to structure
determination.
The CRISPR/Cas9 system has revolutionized many fields of
life sciences by making it possible to modify the genome sequence
with unprecedented efficiency and precision [4, 5]. For example, it
is now possible to insert fusion tags at endogenous loci of mammalian cells, providing an efficient way to undertake affinity purification of macromolecular complexes and/or visualize their
distribution and dynamics in a cellular context [6, 7]. We have
recently detailed a simplified protocol for CRISPR/Cas9-mediated
gene tagging in human cell lines using chemically modified singlestranded oligonucleotides encoding a small affinity tag as donor
template and a co-selection strategy targeting the ATP1A1 gene
[8] (to be published in MiB, Editor R Owens). Here we describe a
procedure based on the use of the CRISPR/Cas9 system and
double-stranded DNA donors for tagging the protein of interest
with a fluorescent reporter and detail the generation of an U2-OS
::
XPB-GFP knocked-in cell line expressing a XPB-GFP fusion protein.
XPB is a subunit of the TFIIH complex, essential in initiation of
DNA transcription by RNA polymerase II and DNA repair by
nucleotide excision repair [9, 10]. This XPB-GFP-tagged U2-OS
cell line was instrumental to establish a partnership between TFIIH
and the histone acetyl transferase GCN5 and the impact of TFIIH
on GCN5 activity with important consequences on gene expression
and chromatin structure [11].
2 Materials
Procedures described here need access to standard equipment for
molecular biology (PCR amplification, agarose gel analysis, bacteria
transformation, access to a DNA sequencing/synthesis service,
etc.), cell culture (cryo-container and liquid nitrogen source, temperature- and CO 2 -controlled incubator, laminar flow hood, centrifuge with adaptor for 15 and 50 mL tubes, cell counter), cell
microscopy (fluorescence microscope with 63Â or higher magnification objectives, etc.), and protein analysis (refrigerated microcentrifuge, a small-scale ultrasonic homogenizer, nano-UV spectrometer, protein gel electrophoresis, and Western blotting transfer
system, etc.). Basic knowledge in these fields is expected.
Experiments detailed below were performed in U2-OS cells
(ATCC HTB-96). These adherent cells are grown at 37
C, 5%
CO 2 in McCoy’s 5a or DMEM-based culture medium (see Note 1).
For experiments in Subheadings 3.2–3.5, in addition to specific
materials, you will need:
40
Sylvain Geny et al.
determination.
The CRISPR/Cas9 system has revolutionized many fields of
life sciences by making it possible to modify the genome sequence
with unprecedented efficiency and precision [4, 5]. For example, it
is now possible to insert fusion tags at endogenous loci of mammalian cells, providing an efficient way to undertake affinity purification of macromolecular complexes and/or visualize their
distribution and dynamics in a cellular context [6, 7]. We have
recently detailed a simplified protocol for CRISPR/Cas9-mediated
gene tagging in human cell lines using chemically modified singlestranded oligonucleotides encoding a small affinity tag as donor
template and a co-selection strategy targeting the ATP1A1 gene
[8] (to be published in MiB, Editor R Owens). Here we describe a
procedure based on the use of the CRISPR/Cas9 system and
double-stranded DNA donors for tagging the protein of interest
with a fluorescent reporter and detail the generation of an U2-OS
::
XPB-GFP knocked-in cell line expressing a XPB-GFP fusion protein.
XPB is a subunit of the TFIIH complex, essential in initiation of
DNA transcription by RNA polymerase II and DNA repair by
nucleotide excision repair [9, 10]. This XPB-GFP-tagged U2-OS
cell line was instrumental to establish a partnership between TFIIH
and the histone acetyl transferase GCN5 and the impact of TFIIH
on GCN5 activity with important consequences on gene expression
and chromatin structure [11].
2 Materials
Procedures described here need access to standard equipment for
molecular biology (PCR amplification, agarose gel analysis, bacteria
transformation, access to a DNA sequencing/synthesis service,
etc.), cell culture (cryo-container and liquid nitrogen source, temperature- and CO 2 -controlled incubator, laminar flow hood, centrifuge with adaptor for 15 and 50 mL tubes, cell counter), cell
microscopy (fluorescence microscope with 63Â or higher magnification objectives, etc.), and protein analysis (refrigerated microcentrifuge, a small-scale ultrasonic homogenizer, nano-UV spectrometer, protein gel electrophoresis, and Western blotting transfer
system, etc.). Basic knowledge in these fields is expected.
Experiments detailed below were performed in U2-OS cells
(ATCC HTB-96). These adherent cells are grown at 37
C, 5%
CO 2 in McCoy’s 5a or DMEM-based culture medium (see Note 1).
For experiments in Subheadings 3.2–3.5, in addition to specific
materials, you will need:
40
Sylvain Geny et al.