Keep A Knockin: new paper using knock-in technology

We have a new paper out! I am a bit late with this post, since the accepted version went online in December 2020, and the final version appeared a few weeks ago. It will shortly appear in a finished issue of the journal so I can tell myself that I am not too late yet.

What’s it about?

A complex of TACC3-chTOG-clathrin-GTSE1 is important for stabilising the mitotic spindle during cell division. We used CRISPR/Cas9 gene-editing to knock-in a tag on each of these proteins. The tag that allowed us to see each protein and to do “endogenous knocksideways”. This meant we could determine how the parts of the complex fit together and how it binds microtubules.

As a bonus, a further protein (PIK3C2A) had been proposed to be part of the complex, and we show that it is not.

Endogenous knocksideways

Knocksideways was developed by Scottie Robinson as a way to inactivate proteins in cells. The method removes the protein from its normal site of action and sticks it to the mitochondria. It’s not a knock-down or a knock-out, it’s a knocksideways – geddit? Knocksideways relies on the addition of an FKBP tag to the protein-of-interest and CRISPR allows us to make sure all of the endogenous protein has this tag.

As well as inactivation, this method allows us to determine which proteins bind to the protein-of-interest.

Knocksideways of protein X means that we can determine proteins that bind to X (such as protein Y1) and ones that don’t (protein Y2)

We used this method to show that TACC3 and clathrin bind each other and they control how the complex binds the spindle microtubules. GTSE1 binds clathrin and chTOG binds TACC3, but they don’t affect microtubule binding of the complex.

GFP-FKBP-TACC3 (green) being removed from the spindle and attached to the mitochondria (purple)

The people

Ellis and James are joint-first authors of the paper. The gene-editing project was initiated by Ellis Ryan, assisted by Teresa Massam-Wu. They built on earlier work getting tagging up-and-running in the lab by Cristina Gutiérrez Caballero. Isolating and characterising knock-in clones with endogenous tagging is very laborious and James Shelford joined the characterisation effort. Ellis and James did the experimental work in the paper once the cell lines were made.

Huge thanks to James who did most of the revisions for the paper while working under COVID-19 restrictions in the lab. The work was supported by a Cancer Research UK programme award in collaboration with Richard Bayliss.

Review Commons experience

This was our first paper to go through Review Commons. For those that do not know, the idea is that authors deposit their preprint and then “submit” it to Review Commons. Review Commons organises peer review of the manuscript. Importantly, the review is journal-agnostic and peers focus on the scientific soundness of the paper although they can also comment on potential impact. With the reviews conducted, the authors can construct a revision plan and talk to prospective journals about publishing the paper.

In our case we got three very constructive reviews with some suggestions for experiments to improve the manuscript. With a straightforward path, we thought we would try a couple of journals, but they declined the paper since it wasn’t novel enough. We were happy for the paper to find a home at J Cell Sci, which is where we should have sent it in the first place!

We posted the work on bioRxiv on 1st July 2020. The review process was a little slower due to the pandemic, but the paper was accepted 14th December and went online on 30th December. Again, kudos to JCS for a rapid process.

The post title comes from “Keep A-Knockin’ (But You Can’t Come In)” as recorded by Little Richard on his “Little Richard” LP. The drum part to this song was famously re-used by John Bonham as the basis for “Rock and Roll” on Led Zeppelin’s fourth album.

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