Here's a thread telling the story of our new technique for genetic recording of lineage history during long term evolution experiments.
The work was co-led with @alex_nguyen_ba and @ivana_cvijovic in @MichaelMDesai's lab.
Read the paper here:http://nature.com/articles/s41586-019-1749-3 …
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Over the next couple of days, I'll describe some of the essential components of our technology and how they make long term lineage tracking possible, including: A novel, hyper-directional, set of Lox sites Reiterative Cre-Lox cassette exchange The splicing trap
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Our system requires that the barcodes we integrate into the genome stay there,even after hundreds of generations of evolution. Unfortunately, Cre-Lox integration is reversible. Worse yet, at equilibrium, integration substrates are favored over integrated product.pic.twitter.com/YMF4vPfrcG
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An ingenious solution to this problem takes advantage of mutant Lox inverted repeats (IRs) thought to decrease Cre binding. Since Cre binding to Lox IRs is cooperative, a site with a single mutated IR will recombine normally but a double mutant site will not.pic.twitter.com/rYtWqqrqIz
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If the product of recombination contains a doubly mutated Lox site, the reaction equilibrium shifts towards integration.pic.twitter.com/FthmNV8CDP
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@alex_nguyen_ba and I designed an in vitro assay to measure the stability of Cre-Lox integration products and tested a large number of inverted repeat mutations from the literature.pic.twitter.com/JK0RBbUJaU
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Only a single arm mutation was able to reduce the rate of excision to acceptable levels (more than 6 orders of magnitude lower than WT) while still allowing integration. It is difficult to understate importance of this result to the viability of our technique.
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The shift from a reaction with an equilibrium that favors integration to a reaction that produces effectively stable integration means the difference between a system that will work by itself and a system that can become a module in a more complex technology.
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