We are so excited to present our functional #lineagetracing tool, CaTCH, developed in the @obenaufa lab @IMPvienna published @naturebiotech. Have you ever wondered if a phenotype is pre-existing in a cell or whether it is acquired during selection? Then👇might be your tool!1/14 
CaTCH - short for “CRISPRa Tracing of Clones in Heterogeneous cell populations” - combines precise mapping of the lineage history of millions of cells with the ability to isolate any given clone alive from a complex population based on genetic #barcodes. 2/14
In a selection experiment, CaTCH allows to first identify clones with a certain phenotype and subsequently “travel back in time” to enable the retrospective isolation and analysis of founding clones from heterogeneous cell populations prior to their evolutionary selection. 3/14
How does it work? Every clone is tagged with a unique barcode (BC) coupled to an inducible GFP reporter. To isolate specific clones, we use CRISPRa with a sgRNA that targets the BC of interest. This recruits dCas9-VPR that induces GFP expression, allowing isolation by FACS.4/14
By spiking in clones with a single barcode and specifically targeting them for activation, we demonstrated that CaTCH has the resolution and sensitivity to detect and isolate specific clones at least as rare as 0.001% within a complexly barcoded population. 5/14
One question we were really intrigued by is whether resistance to therapy is a pre-existing or an acquired feature. Thus, we used CaTCH to provide insights into the evolution of resistance to targeted RAFi/MEKi therapy in an immunocompetent mouse melanoma model. 6/14
First, we generated RAFi/MEKi-resistant tumors in vivo and sequenced their barcodes. We interestingly found that resistant tumors showed a high degree of clonality with only 1-6 clones dominating the tumor. 7/14
Using CaTCH we could successfully isolate clones which had enriched in the resistant tumors. We sorted them from both the resistant (R) population and from the treatment-naïve (TN) starting population, providing us with TN/R clonal pairs. 8/14
When we re-challenged the isolated clones with RAFi/MEKi therapy in vivo, we saw that most clones responded in their TN state before relapse, indicating that they had acquired the resistance. 9/14
In one of the resistant clones, we identified a Kras mutation potentially contributing to the resistant phenotype. However, this mutation was not detected in its TN counterpart, suggesting that this mutation was acquired de novo. 10/14
If you are curious to find out more about CaTCH and learn about the technical optimizations we undertook to generate, please have a look at our paper in @naturebiotech: nature.com/articles/s4158… 11/11.
Also check out our Nature community blog post tiny.cc/zroksz. 11/14
Also check out our Nature community blog post tiny.cc/zroksz. 11/14
A big thank you to all co-authors and collaborators (on Twitter: @holstein_f, @Juli_Bla, @fruce_ki, @fh_fingolfin, @thomaswiesner, @johannes_zuber), the fantastic core-facilities @VBCF_Tweets of @IMPvienna and the @obenaufa lab funding agencies @ERC_Research and @WWTF. 12/14
Many thanks also to referees for their time and their comments that improved our manuscript and @MarkusElsner1 @naturebiotech for the fantastic experience in publishing our first paper of the lab. 13/14
If you would like to try #CaTCH yourself – we will make the plasmids available on @Addgene and are also happy to collaborate! Finally – we are looking for postdocs with a passion for science to join our lab @IMPvienna @viennabiocenter! 14/14
An artistic interpretation of the #CaTCH workflow as a blueprint by the one and only @PhilDexheimer @viennabiocenter #PhDstudentsrock
Idea for the illustration by my twitterless student Chris in discussion with the lab and executed by @lisa_obenauf
