Wednesday, 23 December 2015

Seamless gene deletion

2015 is the year that genome editing really became big news. A new technique, "CRISPR/CAS", was named as Science magazine's breakthrough of the year as voted by the public from a shortlist chosen by staff.

However, people have been manipulating DNA through many useful methods long before CRISPR/CAS made headlines. Gene deletion is an important tool when trying to understand the function of genes. Take out a gene and see what effect it causes. Genes can be disrupted (by removing a portion of the DNA or inserting some extra DNA) or can be interfered with, for example via their RNA production, or they can be entirely deleted. It's common practice when removing a gene to insert a marker, so that we can easily select for the cells where this procedure has been successful. For example, to insert an antibiotic resistance gene as a marker, so that we can now grow the cells on a plate with an antibiotic. Then only those that have lost our gene of interest and gained antibiotic resistance will now grow. The trouble with this is that many gene deletions have no visible effect by themselves. If we also want to delete a second gene and a third, then we need more markers, or we need to be able to remove and reuse the marker we inserted. We also don't want the process to leave any scars behind that could destabilise the genome. We've just published a paper to help solve this problem.

This process of 'swap a gene of interest for a marker gene' can be achieved in many organisms by homologous recombination. This is a process used by many cells to repair broken strands of DNA. If we provide a piece of DNA that has a good region of similarity to the region just downstream of our gene of interest, and also a good region of similarity to the region just upstream of the gene of interest, but instead of the gene of interest, has the marker gene between these regions, then the normal cellular processes of homologous recombination will exchange the two. Some organisms perform homologous recombination very readily (S. cerevisiae for example). Others may need a little more encouragement, such as creating a double stranded break.

Our new paper A tool for Multiple Targeted Genome Deletions that Is Precise, Scar-Free and Suitable for Automation with Wayne Aubrey as first author uses a 3-stage PCR process to synthesise a stretch of DNA (a 'cassette') that will do everything. It will have good regions of similarity to the regions upstream and downstream of the gene of interest. It will contain a marker gene. And (here's the good bit), it will contain a specially designed region ('R') before the marker gene that is identical to the region that occurs just after the gene of interest. In this way, after homologous recombination has done its thing and inserted the DNA cassette instead of the gene of interest, there will be two identical R regions, one before the marker gene, and one after the marker gene. Sometimes the DNA will loop round on itself, the two R regions will match up and homologous recombination will snip out the loop, including the marker gene.

We can encourage this to happen and select for the cells that have had this happen if our marker is also 'counter-selectable'. That is, we'd like a marker for which we can add something to the growth medium so that now only cells without the marker will now grow. That is, we'd like to use a marker or marker combination for which we can first select for its presence and then counter-select for its absence. When we have this we can select for cells that have had the marker replace the gene, and then counter-select for cells that have now lost the marker too. So we have a clean gene deletion.

Of course we're always standing on the shoulders of giants when we do science. Our method is an improvement on a method by Akada 2006, so that no extra bases are lost or gained and the method requires no gel purification steps. Just throw in your primers and products and away you go. It's not fussy about quantity. No purification steps means that it could be automated on lab robots. And it could be used to delete any genetic component, not just genes. Give it a try!

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