Still Using ChIP Seq? 
Try CUT&RUN for Enhanced Chromatin Profiling

July 23rd, 2019
Guest blogger | Kelsey Noll
Studying mechanisms of host genetic regulation of antibody response to Influenza A Virus
Ph.D. Student in the Department of Microbiology and Immunology at UNC

Training Initiatives in Biomedical & Biological Sciences | TIBBS Fellow

Immersion Program to Advance Career Training | ImPACT

Chromatin ImmunoPrecipitation (ChIP), the most widely used method for assaying protein-DNA interactions, suffers from a number of limitations that limit its accuracy and sensitivity. The standard ChIP assay is time intensive, has fairly low resolution, requires a large input of cells, and is incompatible with insoluble chromatin proteins. 

The Cleavage Under Targets and Release Using Nuclease (CUT&RUN) approach, developed by Skene and Henikoff, evolved from Chromatin ImmunoCleavage (ChIC)12. By adapting the original method with a fusion of Proteins A and G to micrococcal nuclease  (pAG-MNase),  CUT&RUN drastically decreases background, making it amenable to reduced cellular input and lower read depths compared to other chromatin mapping methods.

"CUT&RUN is an attractive method that is rapidly gaining traction as it vastly outperforms ChIP, the current gold-standard genomic mapping assay"

Comparison of ChIP, ChEC, ChIC, and CUT&RUN chromatin profiling methods

During ChIP, a pool of fragmented chromatin is treated with an antibody specific to a chromatin-associated protein or histone PTM. Isolated DNA associated with the antibody target can be purified and prepared for targeted (ChIP-qPCR) or genome-wide (ChIP-Seq) analysis.

In 2004, the Laemmli group pioneered two novel methods to study protein-DNA interactions, Chromatin Endogenous Cleavage (ChEC) and ChIC. These protocols were cleverly designed to eliminate the initial chromatin fractionation and solubilization steps required in conventional ChIP, via the use of a modified micrococcal nuclease (MNase) that specifically cleaves DNA at regions interacting with a protein of interest
3. Importantly, MNase is activated only in the presence of Ca2+ ions, allowing for controlled activation of the cleavage step. In ChEC, which can be performed on either native or fixed cells, the protein of interest is genetically modified to include an MNase domain. In ChIC, the protein of interested is first tagged by an antibody. The antibody is then recognized by Protein A, which is fused to the MNase. The ChIC method of tethering the MNase via Protein A does not require any transgenic modification to the target protein and can be adapted for most ChIP studies. Both ChIC and ChEC were shown to be highly specific and have up to 10x higher resolution than conventional ChIP methods3,4.

With CUT&RUN, cells (or nuclei) are immobilized on lectin-coated magnetic beads, permeabilized, and incubated with an antibody to a chromatin target (e.g. histone PTM or chromatin / DNA binding protein). Next, pAG-MNase is added and activated via Ca2+. The clipped chromatin fragments diffuse, followed by DNA purification and next-generation sequencing. 

"CUT&RUN uses fewer cells at a lower cost, yet generates data with better resolution and lower background"

Key features of genomic mapping approaches

 Researchers desperately need better methods to advance the study of chromatin and improve its application towards drug development and personalized medicine. Despite its omnipresence, ChIP suffers from a number of serious drawbacks that limit its accuracy and sensitivity. Modifications to the protocol (e.g. ChIP-exo, which utilizes an exonuclease to achieve higher resolution) address some, but not all, of the issues5. Combined with its high resolution and a dramatically accelerated workflow, CUT&RUN is an attractive method that is rapidly gaining traction as it vastly outperforms those using ChIP, the current gold-standard genomic mapping assay.

"CUT&RUN has been applied to various projects, a number of which were made possible only because of advances this assay offers compared to conventional protein-DNA interaction mapping methods"

CUT&RUN in scientific literature

CUT&RUN has been applied to various projects, including regulation of developmental gene programming6,7,8, analysis of CRISPR-modified transcription factor binding9, maternal imprinting10, and cell cycle regulation11,12. Notably, a number of these studies were made possible only because of advances in CUT&RUN compared to conventional protein-DNA interaction mapping methods.

Additional modifications to the CUT&RUN protocol have expanded its application to more challenging targets, such as large or insoluble protein complexes2. CUT&RUN has been combined with salt fractionation to study centromeric chromatin (CUT&RUN.Salt)13, and with ChIP to study chromatin factor co-occupancies (CUT&RUN.ChIP)14. It has also been modified to isolate chromatin-associated RNA (CUT&RUNER)15, and to work with ultra-low inputs such as single cells and pre-implantation embryos (uliCUT&RUN)16.

The latest modifications to the standard CUT&RUN method further improve upon the base methodology. Specifically, the Henikoff and Ahmad groups have developed a hybrid Protein A-Protein G-MNase construct to expand antibody compatibility, a modified digestion protocol, a novel-peak calling strategy, and a calibration strategy using carryover bacterial DNA17.

There has been a rapid and growing interest in CUT&RUN since its publication in 2017. The protocol has been made available on an open source repository where it has received over 20,000 views (second only to a recipe for 50X TAE in popularity), and materials have been distributed to over 500 laboratories world-wide. Currently, there are 27 papers published or in preprint that utilize CUT&RUN or a variant thereof, with almost half of those in 2019 alone so far, highlighting the rapid adoption and excitement surrounding this novel method. CUT&RUN promises to be a groundbreaking strategy for the study of protein-DNA interactions, producing high quality data from low cell numbers in a fast, approachable manner.

Interested in trying CUT&RUN?

 EpiCypher®  has recently licensed ChIC / CUT&RUN technologies and will soon be releasing CUTANA™ assays, the first commercial products for CUT&RUN.  Contact EpiCypher's team of scientists with any questions about assay setup and optimization! In additon, Dr. Henikoff's lab maintains an active online forum for the CUT&RUN protocol, serving as an important technical resource.

References

1. Skene PJ, et al. Targeted in situ genome-wide profiling with high efficiency for low cell numbers. Nat Protoc, 2018. 13(5): p. 1006-19. (PubMed PMID: 29651053)

2. Skene PJ, Henikoff S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. Elife, 2017. 6: p. (PubMed PMID: 28079019) (PMC5310842)

3. Schmid M, et al. ChIC and ChEC; genomic mapping of chromatin proteins. Mol Cell, 2004. 16(1): p. 147-57. (PubMed PMID: 15469830)

4. Orlando V. Mapping chromosomal proteins in vivo by formaldehyde-crosslinked-chromatin immunoprecipitation. Trends Biochem Sci, 2000. 25(3): p. 99-104. (PubMed PMID: 10694875)

5. Rhee HS, Pugh BF. Comprehensive genome-wide protein-DNA interactions detected at single-nucleotide resolution. Cell, 2011. 147(6): p. 1408-19. (PubMed PMID: 22153082) (PMC3243364)

6. Liu N, et al. Direct Promoter Repression by BCL11A Controls the Fetal to Adult Hemoglobin Switch. Cell, 2018. 173(2): p. 430-42 e17. (PubMed PMID: 29606353) (PMC5889339)

7. Uyehara CM, McKay DJ. Direct and widespread role for the nuclear receptor EcR in mediating the response to ecdysone in Drosophila. Proc Natl Acad Sci U S A, 2019. 116(20): p. 9893-902. (PubMed PMID: 31019084) (PMC6525475)

8. Zheng XY, Gehring M. Low-input chromatin profiling in Arabidopsis endosperm using CUT&RUN. Plant Reprod, 2019. 32(1): p. 63-75. (PubMed PMID: 30719569)

9. Roth TL, et al. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature, 2018. 559(7714): p. 405-9. (PubMed PMID: 29995861) (PMC6239417)

10. Inoue A, et al. Maternal Eed knockout causes loss of H3K27me3 imprinting and random X inactivation in the extraembryonic cells. Genes Dev, 2018. 32(23-24): p. 1525-36. (PubMed PMID: 30463900) (PMC6295166)

11. Park SM, et al. IKZF2 Drives Leukemia Stem Cell Self-Renewal and Inhibits Myeloid Differentiation. Cell Stem Cell, 2019. 24(1): p. 153-65 e7. (PubMed PMID: 30472158) (PMC6602096)

12. Albert B, et al. Sfp1 regulates transcriptional networks driving cell growth and division through multiple promoter-binding modes. Genes Dev, 2019. 33(5-6): p. 288-93. (PubMed PMID: 30804227) (PMC6411004)

13. Thakur J, Henikoff S. Unexpected conformational variations of the human centromeric chromatin complex. Genes Dev, 2018. 32(1): p. 20-5. (PubMed PMID: 29386331) (PMC5828391)

14. Brahma S, Henikoff S. RSC-Associated Subnucleosomes Define MNase-Sensitive Promoters in Yeast. Mol Cell, 2019. 73(2): p. 238-49 e3. (PubMed PMID: 30554944) (PMC6475595)

15. Daneshvar K, et al. lncRNA DIGIT and BRD3 protein form phase-separated condensates to regulate endoderm differentiation. bioRxiv, 2019. p.)

16. Hainer SJ, et al. Profiling of Pluripotency Factors in Single Cells and Early Embryos. Cell, 2019. 177(5): p. 1319-29 e11. (PubMed PMID: 30955888) (PMC6525046)

17. Meers MP, et al. Improved CUT&RUN chromatin profiling tools. Elife, 2019. 8: p. (PubMed PMID: 31232687) (PMC6598765)