How CUT&RUN completely changed the chromatin profiling game
Author: Derek Janssens
Derek Janssens is a member of the Henikoff Lab at the Fred Hutchinson Cancer Research Center. In this guest blog post, Derek discusses the CUT&RUN method in detail and how his lab uses protocols.io to enable discussions and feedback.
Since it’s conception three years ago, the CUT&RUN method has been steadily gaining steam and is slowly replacing ChIP-seq as the go to method for chromatin profiling. Now that CUT&RUN has become a household name in biology labs around the world it is easy to forget just how revolutionary the idea initially was. To put it in context, one of the last experiments I completed as part of my graduate thesis was to perform ChIP-seq on the brains of fruit fly larvae. I’ll spare you the details, but this experiment required myself and an undergraduate student — when I was able to convince him to help — to dissect thousands of fly brains that I then processed over the course of several days under the supervision of the resident ChIP-seq expert. After several painstaking months of this, we were able to optimize the protocol enough to complete the experiment, allowing me to put the finishing touches on my thesis and graduate. So, imagine my amazement when, just a few months later, Steve Henikoff described the CUT&RUN technique to me. Using CUT&RUN, we could have generated similar data using less than 100 fly brains and finished the experiment in less than a week. It makes those days of dissecting thousands of fly brains seem almost crazy in hindsight, but I guess that is just the nature of new technology.
What makes CUT&RUN so different? Much like ChIP-seq, CUT&RUN uses an antibody to target the protein of interest, and this preserves the versatility of the method, so that CUT&RUN can in theory be used to profile the genome-wide occupancy of any protein as long as an antibody is available. The primary difference is that while ChIP-seq relies on sonication to indiscriminately lyse cells and break up the protein-DNA complexes into soluble pieces, CUT&RUN is performed with the cells still largely intact, and uses the antibody to tether a pA-MNase fusion protein –the key ingredient of CUT&RUN– in place to locally digest and solubilize only the DNA of interest. This fundamental difference not only simplifies the workflow and makes experiments more consistent, it also dramatically reduces the amount of non-specific DNA that ends up getting sequenced. As Steve puts it, “In ChIP-seq you are pulling a needle out of a haystack; in CUT&RUN you leave the haystack behind.” The end result is less background, meaning less starting material is required to get high quality data, and Voila! an experiment that would have been a major undertaking for a lab can now be done by a single student in less than a week to produce even better data. It has been a real game changer in the field of chromatin profiling.
Once we showed this fundamentally different approach to chromatin profiling not only works, but in many cases, produces better data, the race was on, and a full out triathlon of technological innovation ensued. In the following year, we developed CUT&RUN.Salt, making the method compatible with low solubility complexes such as those bound to the centromere. This was followed in short order by CUT&RUN.ChIP, to look at the co-occupancy of multiple proteins on the DNA, as well as AutoCUT&RUN which dramatically increases the throughput, allowing a single operator to generate up to 96 CUT&RUN libraries in two days. Then another significant technological leap forward was made with the development of CUT&Tag. Here, the key ingredient of CUT&RUN, pA-MNase, is swapped out for pA-Tn5, an antibody tethered transposase. Not only does this modification further reduce the background and simplify the workflow, but it also makes CUT&Tag compatible with a number of single cell profiling platforms. In fact, earlier this month two preprints — one from our group, and one from the Karolinska Institutet– were posted to bioRxiv, demonstrating the utility of CUT&Tag for profiling the heterogeneity of the repressed chromatin landscapes of single human blood and brain cells on the 10X Genomics microfluidics platform. The combined accessibility of the 10X platform and versatility of CUT&Tag now make profiling specific chromatin associated proteins an integral part of the single cell genomics revolution that is underway.
In addition to the versatility of the methods themselves, tireless community outreach efforts have also contributed to the growing popularity of CUT&RUN and CUT&Tag. For example, before the CUT&RUN and CUT&Tag reagents were made commercially available, our lab manager, Terri Bryson led the charge to mail aliquots of pA-MNase and pA-Tn5 to hundreds of labs around the world. This culminated in a day were the entire lab worked as an assembly line to put together several hundred CUT&Tag “care packages.” Another critical component of this community outreach is made possible by the protocols.io website for methods sharing. In fact, the CUT&RUN protocol was one of the first to be posted on protocols.io, and this page now enjoys several thousand users a month. Several features of protocols.io have made the platform invaluable to our lab. A discussion forum is built into each of the protocols, allowing users to ask questions and provide feedback about specific steps. We could not have anticipated the variety of ways people would apply these methods, and this discussion forum allows us to help users troubleshoot in real time, while allowing others to take advantage of the discussion for their own experiments. Another attractive feature of protocols.io is that it allows for periodic updates to the protocols. This way, when we identify useful modifications in the course of our experiments, we can easily keep the community apprised on the state of the art. Currently, the CUT&RUN protocol is on its third iteration, and now provides a variety of options to meet the needs of different users.
As is often the case, doing good work results in getting more work to do. After I provided useful feedback to several users, Steve asked me to take the lead on maintaining the CUT&RUN protocols.io page, and I also maintain the AutoCUT&RUN protocols.io page to help users with high-throughput needs get AutoCUT&RUN set up at their institute. This has been a very successful model at Fred Hutch, and AutoCUT&RUN is currently offered as a core service through the genomics shared resources facility. In conjunction with the publication of the CUT&Tag method, Steve set up a CUT&Tag protocols.io page that he continues to maintain. For new users interested in chromatin profiling, these sites are a great place to start. We no longer provide the reagents directly, but both pA/G-MNase and pA/G-Tn5 can be purchased from Epicypher, or alternatively new users can obtain the pA/G-MNase or pA-Tn5 plasmids from Addgene and prepare their own stock reagents by following the purification protocol.
So, what’s on the horizon for chromatin profiling in the Henikoff Lab? Recently, we modified the CUT&Tag workflow to produce a streamlined protocol we are calling CUT&Tag Direct. This strategy makes the method so simple that during the Covid-19 shutdown several members of our lab, including Steve, started running their profiling experiments at home. To help other users continue their research during the pandemic, Steve set up a new protocols.io page for CUT&Tag@home. By taking advantage of the protocol modifications that enable Tn5 release and PCR in the same sample reaction chamber, we adapted CUT&Tag to an automated 96 well format, and made the AutoCUT&Tag protocol available on protocols.io. By using AutoCUT&Tag in combination with single cell CUT&Tag profiling we are exploring the regulatory heterogeneity in acute pediatric leukemia samples to understand how related oncogenic translocations in the mixed lineage leukemia-1 gene give rise to different phenotypic outcomes and varying degrees of tumor plasticity. The initial findings of this project are now publicly available as a preprint on bioRxiv, and we are excited about the prospect of integrating these methods with clinical trials to use chromatin profiling as a diagnostic readout. In addition, several exciting tech-dev projects are underway in the lab that use modifications to the CUT&Tag method to explore novel aspects of the chromatin landscape, so stay tuned as we hope to release these exciting new developments by the end of the year.