CRISPR is for genome engineering what Mike Tyson was for boxing: simple, efficient and almost a guarantee to see a knockout in the first round. While in boxing a knockout typically results in an involuntary power nap of the opponent, the phenotype of a gene knockout is often not so clear-cut. Besides potential problems caused by redundancy and lethality, additional information about expression patterns, protein interaction partners and subcellular localization is required to truly understand gene function.
Spurred on by this challenge, an inventive group of researchers from the Ludwig-Maximilians University in Munich developed an all-in-one solution for systematic gene functional studies using genome engineering. Pairing up CRISPR with the serine integrase Bxb1, the researchers developed a multifunctional integrase (MIN) tag that doubles as a genetic entry site and as a novel epitope tag.
Here is how it works:
- The MIN-tag consists of a 48 bp-long Bxb1 phage attachment site (attP).
- Using CRISPR-assisted targeting the MIN-tag is inserted into the open reading frame of a target gene.
- The MIN-tag is inserted either directly downstream of the start codon (N-terminal tagging) or directly upstream of the stop codon (C-terminal tagging) .
- Once inserted, the MIN-tag is translated into a novel epitope tag that can be detected with a specific antibody.
- At the same time the MIN-tag can be used to insert different prefabricated cassettes into the locus by Bxb1-mediated recombination.
- Bxb1-mediated recombination is highly efficient (50–70%), which allows fast and simple generation of isogenic cell lines with different functionalizations such as GFP knock-ins, cDNA knock-ins or enzymatic tags.
The researchers go ahead and tag all major genes involved in DNA modification including Tet1, Tet2 and Tet3 as well as the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b in mouse embryonic stem cells. They show that the antibody directed against the MIN peptide can be used for immunoprecipitation and immunofluorescence experiments in different MIN-tagged cell lines, eliminating the need to generate gene specific antibodies.
The authors also create a toolbox of plasmids that can be used for functionalization of MIN-tagged cell lines by Bxb1-mediated recombination. The researchers demonstrate the versatility of their approach by identifying novel interactors of TET1 by proximity-dependent protein labeling and by uncovering the spatio-temporal dynamics of DNMT3B and its isoforms during epiblast differentiation.
Lead author Christopher Mulholland points out that “Just in our hands, we have been able to address previously intractable biological questions due to the high efficiency and flexibility of the MIN-tag system. Additionally, due to the universal compatibility of the toolbox constructs, we could use the same prefabricated plasmids to modify several genes and all that in record time.”
To make the system easily accessible to the scientific community the researcher set up MINtool, an online platform for targeting strategy design and the distribution of plasmids, cell lines as well as the antibody.
Learn how to MINimize your genome engineering efforts and head over to Nucleic Acids Research, May 2015.