Julia Fueller, Konrad Herbst, Matthias Meurer, Krisztina Gubicza, Bahtiyar Kurtulmus, Julia D. Knopf,
Daniel Kirrmaier, Benjamin Buchmuller, Gislene Pereira, Marius K. Lemberg, Michael Knop
PCR tagging is a one-step procedure for chromosomal gene tagging in mammals
PCR tagging enables the rapid creation of cell lines with targeted large chromosomal insertions, such as GFP
How does PCR tagging work?
a gene specific PCR cassette is transfected into the target cells together with a helper plasmid containing a Cas12a endonuclease
the insertion of the PCR cassette into the chromosome yields a fusion of the tag (e.g. GFP) with the target gene
How is the PCR cassette generated?
two gene specific tagging oligos (M1 and M2) provide the homology arms (50 to 90 nts in length) for targeted integration by HDR
the M2 tagging oligo additionally provides a protospacer sequence for the Cas12a endonuclease
the generic template cassette provides the tag (e.g. a fluorescent protein) and additional features (e.g. a selection marker)
it also contains the backbone of a Cas12a specific crRNA gene, consisting of promoter and crRNA direct repeat
What is the tagging principle?
the PCR cassette contains a crRNA gene that is expressed inside the cell
the crRNA directs Cas12a (expressed from the helper plasmid) to the target locus close to the insertion site
stimulated by the double strand break the linear tagging cassette is inserted into the genome by HDR
the homology arm of the M1 tagging oligo directs in frame fusion of the tag with the target ORF,
leading to the expression of a tagged protein from the target locus
integration leads to destruction of the crRNA target site, thus preventing re-cleavage of the locus
What do I need for PCR tagging?
the sequence of the target locus (e.g. your favorite gene)
*HiFi-Polymerase is our self-purified DNA polymerase
10x HiFi buffer - to be used with any polymerase
200 mM Tris-HCl, pH 8.8
100 mM (NH₄)₂SO₄
500 mM KCl
1% (v/v) Triton X-100
1 mg/ml BSA
20 mM MgCl₂
We note that the Phusion polymerase using the manufacturer supplied buffer does not work for cassette amplification with M1 and M2 tagging oligos,
whereas for Velocity polymerase using the buffer provided by the manufacturer good amounts of the product are obtained.
We suggest pipetting on ice and pre-heating the PCR machine or hot start.
We found that all polymerases work well using the buffer conditions and amplification above.
Julia Fueller, Konrad Herbst, Matthias Meurer, Krisztina Gubicza, Bahtiyar Kurtulmus, Julia D. Knopf,
Daniel Kirrmaier, Benjamin Buchmuller, Gislene Pereira, Marius K. Lemberg, Michael Knop
The information contained in this website is correct to the best of our knowledge.
Under no circumstance shall the authors be liable for any potential mistakes, claim, damage or loss arising from the use of the application
'Online oligo design tool for PCR tagging in mammalian cells'.
The use of the tool and the reliance on any information on the site is solely at the user's own risk.
Only for non-commercial, academic or research purposes.
Updates
21.06.2022 plasmids.eu links added to template list