5. With the XPB-FW2/XPB-RV2 primers which hybridize on
either side of the insertion site, both knock-in and
non-modified alleles should be amplified: A 2800 bp PCR
product is expected if the eGFP-P2A-PURO was successfully
inserted while a 1300 bp fragment should be observed for a
non-modified allele, allowing to discriminate between homozygous, heterozygous, and non-modified clones. With the
XPB-FW/GFP-RV and Puro-FW/XPB-RV2 pairs which
amplify 540 bp and 1000 bp fragments only modified alleles
will be detected as the GFP-RV and Puro-RV2 oligonucleotides are complementary to the eGFP-P2A-PURO cDNA from
the inserted sequence.
Acknowledgments
We thank IGBMC cell culture and imaging facilities for assistance
and fruitful discussions. This work was supported by the Centre
National pour la Recherche Scientifique (CNRS), the Institut
National de la Sante ´ et de la Recherche Me ´dicale (INSERM),
Universite ´ de Strasbourg (UdS), Association pour la Recherche
sur le Cancer (ARC), the Ligue nationale contre le cancer, Institut
National du Cancer (INCa; INCA 9378), Agence National pour la
Recherche (ANR-12-BSV8-0015-01 and ANR-10-LABX-0030INRT under the program Investissements d’Avenir ANR-10IDEX-0002-02), Instruct (R&D Project Funding), and InstructULTRA as part of the European Union’s Horizon 2020 (Grant ID
731005) by Instruct-ERIC.
References
1. Villicana C, Cruz G, Zurita M (2014) The
basal transcription machinery as a target for
cancer therapy. Cancer Cell Int 14(1):18
2. Scott DE, Bayly AR, Abell C, Skidmore J
(2016) Small molecules, big targets: drug discovery faces the protein-protein interaction
challenge. Nat Rev Drug Discov 15
(8):533–550
3. Bushweller JH (2019) Targeting transcription
factors in cancer—from undruggable to reality.
Nat Rev Cancer 19(11):611–624
4. Doudna JA, Charpentier E (2014) Genome
editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346
(6213):1258096
5. Jinek M, Chylinski K, Fonfara I, Hauer M,
Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease
in adaptive bacterial immunity. Science 337
(6096):816–821
6. Dalvai M, Loehr J, Jacquet K, Huard CC,
Roques C, Herst P, Cote J, Doyon Y (2015)
A scalable genome-editing-based approach for
mapping multiprotein complexes in human
cells. Cell Rep 13(3):621–633
7. Dewari PS, Southgate B, McCarten K,
Monogarov G, O’Duibhir E, Quinn N,
Tyrer
A,
Leitner
MC,
Plumb
C,
Kalantzaki M, Blin C, Finch R, Bressan RB,
Morrison G, Jacobi AM, Behlke MA, von
Kriegsheim A, Tomlinson S, Krijgsveld J, Pollard SM (2018) An efficient and scalable pipeline for epitope tagging in mammalian stem
cells using Cas9 ribonucleoprotein. elife 7
8. Geny S, Pichard S, Poterszman A, Concordet J
(in press) Gene tagging with the CRISPR-Cas9
system to facilitate macromolecular complex
purification. Methods Mol Biol
56
Sylvain Geny et al.
either side of the insertion site, both knock-in and
non-modified alleles should be amplified: A 2800 bp PCR
product is expected if the eGFP-P2A-PURO was successfully
inserted while a 1300 bp fragment should be observed for a
non-modified allele, allowing to discriminate between homozygous, heterozygous, and non-modified clones. With the
XPB-FW/GFP-RV and Puro-FW/XPB-RV2 pairs which
amplify 540 bp and 1000 bp fragments only modified alleles
will be detected as the GFP-RV and Puro-RV2 oligonucleotides are complementary to the eGFP-P2A-PURO cDNA from
the inserted sequence.
Acknowledgments
We thank IGBMC cell culture and imaging facilities for assistance
and fruitful discussions. This work was supported by the Centre
National pour la Recherche Scientifique (CNRS), the Institut
National de la Sante ´ et de la Recherche Me ´dicale (INSERM),
Universite ´ de Strasbourg (UdS), Association pour la Recherche
sur le Cancer (ARC), the Ligue nationale contre le cancer, Institut
National du Cancer (INCa; INCA 9378), Agence National pour la
Recherche (ANR-12-BSV8-0015-01 and ANR-10-LABX-0030INRT under the program Investissements d’Avenir ANR-10IDEX-0002-02), Instruct (R&D Project Funding), and InstructULTRA as part of the European Union’s Horizon 2020 (Grant ID
731005) by Instruct-ERIC.
References
1. Villicana C, Cruz G, Zurita M (2014) The
basal transcription machinery as a target for
cancer therapy. Cancer Cell Int 14(1):18
2. Scott DE, Bayly AR, Abell C, Skidmore J
(2016) Small molecules, big targets: drug discovery faces the protein-protein interaction
challenge. Nat Rev Drug Discov 15
(8):533–550
3. Bushweller JH (2019) Targeting transcription
factors in cancer—from undruggable to reality.
Nat Rev Cancer 19(11):611–624
4. Doudna JA, Charpentier E (2014) Genome
editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346
(6213):1258096
5. Jinek M, Chylinski K, Fonfara I, Hauer M,
Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease
in adaptive bacterial immunity. Science 337
(6096):816–821
6. Dalvai M, Loehr J, Jacquet K, Huard CC,
Roques C, Herst P, Cote J, Doyon Y (2015)
A scalable genome-editing-based approach for
mapping multiprotein complexes in human
cells. Cell Rep 13(3):621–633
7. Dewari PS, Southgate B, McCarten K,
Monogarov G, O’Duibhir E, Quinn N,
Tyrer
A,
Leitner
MC,
Plumb
C,
Kalantzaki M, Blin C, Finch R, Bressan RB,
Morrison G, Jacobi AM, Behlke MA, von
Kriegsheim A, Tomlinson S, Krijgsveld J, Pollard SM (2018) An efficient and scalable pipeline for epitope tagging in mammalian stem
cells using Cas9 ribonucleoprotein. elife 7
8. Geny S, Pichard S, Poterszman A, Concordet J
(in press) Gene tagging with the CRISPR-Cas9
system to facilitate macromolecular complex
purification. Methods Mol Biol
56
Sylvain Geny et al.