CRISPR-mediated Chromatin Immunoprecipitation (Part 54- CRISPR in Gene Editing and Beyond)

Roohi Bansal
Biotechnology by TSB
9 min readOct 23, 2023

Welcome to the 54th part of the multi-part series on applications of CRISPR in gene editing and beyond.

Introduction to ChIP

Chromatin Immunoprecipitation, abbreviated as ChIP, is an antibody-based technology used to determine in vivo interactions occurring between DNA and proteins in the cell by immunoprecipitating chromatin with specific antibodies. The protein can be histones or regulatory proteins such as transcription factors, proteins binding to enhancers, etc. DNA-protein interactions play a key role in regulating several cellular functions, including DNA replication and recombination, DNA repair, gene transcription, chromosomal stability, cell cycle progression, and epigenetic silencing.

Crosslinking: The first step of ChIP assays begins with the covalent stabilization of protein-DNA complexes by crosslinking (Fig 1a). Crosslinking is normally achieved by treating intact cells with formaldehyde.

Cell lysis: In the ChIP protocol, the second step involves extracting chromatin (Fig 1b). This can be achieved by either using a detergent-based solution to dissolve the cell membrane or by using glass beads to disrupt the membrane through bead beating. During the ChIP procedure, the stability of the protein-DNA complex remains unaffected by the presence of detergents or bead beating. This is because the covalent crosslinking maintains the stability of the complex throughout the process.

Fig 1: Chromatin Immunoprecipitation (ChIP) assay

DNA fragmentation: In order to analyze the sequences where proteins bind, it is necessary to fragment the extracted genomic DNA into smaller pieces (Fig 1c). This fragmentation can be achieved by either mechanical methods, such as sonication, or enzymatic digestion using micrococcal nuclease.

Immunoprecipitation: The subsequent step in immunoprecipitation involves selectively enriching the desired protein-DNA complex and removing any other irrelevant cellular material. This is achieved by incubating the sonicated chromatin with antibodies that recognize the target protein and form an antibody-protein-DNA complex (Fig 1d).

However, there are cases where specific antibodies against the target protein are not available. In such cases, fusion proteins tagged with affinity tags such as GST or FLAG can be used instead. To utilize this approach, the fusion protein is expressed in the cells of interest. After crosslinking and sonication, antibodies against the affinity tags are used to immunoprecipitate the target protein. Once the antibody-protein-DNA complex is formed, it is purified using an antibody-binding resin such as immobilized protein A, protein G, or protein A/G (Fig 2). These resins selectively bind to the antibody portion of the complex and allow for the removal of any unbound material.

The choice of resin used depends on the antibody class or subclass used for immunoprecipitation. For example, protein A resin binds to the Fc region of the IgG antibodies, while protein G resin can bind to the Fc regions of IgG, IgM, and IgA antibodies. Protein A/G resin combines the binding properties of both protein A and protein G, allowing for a wider range of antibody classes and subclasses to be captured.

If the target protein is biotinylated, immobilized streptavidin can be used instead of antibody-binding resins.

Fig 2: Affinity purification of DNA-bound tagged proteins-antibody complex using immobilized protein A, G, or A/G

Reverse cross-linking: Subsequently, the crosslinks between DNA and protein are then reversed by either extensive heat incubations and/or by digesting the protein component using proteinase K (Fig 1e). The heat treatment breaks the covalent bonds between the protein and DNA, while proteinase K cleaves peptide bonds in the protein components, both of which help to release the DNA from the protein-DNA complex. Next, the DNA is extracted from the complex with phenol-chloroform, followed by precipitation using ethanol. Phenol-chloroform is a mixture of two organic solvents that can separate DNA from other contaminants such as proteins, lipids, and carbohydrates. Ethanol precipitation is a process where the DNA is mixed with ethanol, which causes the DNA to clump together and form a visible pellet. The pellet is then washed with ethanol to remove any remaining contaminants, and the resulting purified DNA is resuspended in a buffer solution for further analysis.

After the DNA is purified from the complex, the next step in the ChIP process is to examine the in vivo recruitment of the target protein. This is done by using PCR amplification to detect specific DNA sequences that are bound to the protein of interest, such as promoter sequences (Fig 1f). PCR, or polymerase chain reaction, is a powerful technique that can amplify small amounts of DNA into millions of copies. To perform the PCR, primers are designed to amplify the region of interest, which could be a promoter sequence or another region of DNA that is known to interact with the protein of interest. The purified DNA sample is then used as a template for the PCR reaction, along with the appropriate primers and other necessary components like buffer, polymerase enzyme, and dNTPs.

The enrichment of the PCR signal from the immunoprecipitated sample is compared to the signal from the mock sample with no antibody control. The mock sample is a negative control used to determine the background level of DNA amplification in the absence of the protein of interest. By comparing the signal from the immunoprecipitated sample to the mock sample, the region-specific recruitment of the protein can be determined.

If the immunoprecipitation was successful and specific to the protein of interest, there should be a higher level of amplification in the immunoprecipitated sample compared to the mock sample. This difference in amplification signals indicates that the protein is specifically bound to the region of interest in the genome.

By detecting specific DNA sequences bound to the protein of interest, the results obtained from PCR amplification can provide insights into the biological function of the protein and its role in various cellular processes. This information is crucial in understanding the complex mechanisms involved in gene regulation and other biological pathways.

enChIP: CRISPR-mediated ChIP

Using CRISPR, the researchers expanded chromatin immunoprecipitation (ChIP) to identify proteins that are associated with a genomic sequence specified by a particular gRNA (Fujita and Fujii, 2013). This technique is also called engineered DNA-binding molecule-mediated ChIP, abbreviated as enChip. In the enChIP system, catalytically inactive dCas9 is used, which is fused to tags like FLAG (Fujita et al., 2018; Hamidian et al., 2018). A guide RNA (gRNA) is designed to specifically recognize and bind to the target genomic sequence, such as a promoter or other regions of interest. The gRNA then recruits FLAG-tagged dCas9 to a locus of interest, where it forms a complex with the DNA (Fig 3 a and b).

Fig 3: Recruitment of FLAG-tagged dCas9 to target DNA using gRNA

After which, the chromatin is crosslinked to fix the DNA-protein complexes in place (Fig 4a), fragmented (Fig 4b), and dCas9-bound DNA-protein complexes are immunoprecipitated via FLAG-tag specific antibodies, which bind to the FLAG tag on the dCas9 protein (Fig 4c).

Fig 4: Immunoprecipitation of dCas9-bound DNA-protein complexes

Subsequently, the FLAG antibody-dCas9-DNA complex is purified using an antibody-binding resin/beads such as immobilized proteins A, G, or A/G. The crosslinks are then reversed to release the DNA and protein components from the complex (Fig 5a).

The isolated complexes are then analyzed by mass spectrometry to identify the proteins associated with the target DNA sequence (Fig 5b).

Fig 5: Analysis of proteins associated with the target DNA sequence

Mass spectrometry is a powerful analytical technique used to identify and quantify molecules based on their mass-to-charge ratio (m/z). In the context of enChIP, mass spectrometry is used to identify the proteins that are bound to the target DNA sequence. After the immunoprecipitation step, the protein-DNA complexes are isolated and then subjected to protease digestion, which cleaves the proteins into smaller peptides. The resulting peptides are then separated by liquid chromatography based on their chemical and physical properties, such as size, charge, and affinity. The separated peptides are then introduced into the mass spectrometer, where they are ionized, i.e., given a positive or negative charge by applying a high voltage or using a laser. Using an electric or magnetic field, the resulting ions are separated based on their mass-to-charge ratio (m/z). The lighter ions will be deflected more than the heavier ones, allowing for separation based on their m/z ratio. The detector then records the intensities of the ions, producing a mass spectrum that represents the peptides present in the sample.

The mass spectra are then analyzed using database searching software that matches the observed mass spectra to theoretical spectra of known proteins. The software generates a list of proteins that are present in the sample based on the peptide masses and their fragmentation patterns.

The identified proteins are then further validated using additional experimental techniques such as Western blotting or co-immunoprecipitation to confirm their association with the target DNA sequence. Thus, mass spectrometry is a highly sensitive and powerful tool for identifying the proteins associated with a particular genomic region in the enChIP technique.

Tagging dCas9 with BirA: Another approach to tagging dCas9 is by fusing dCAs9 to the biotin ligase BirA (Schmidtmann et al., 2016; Liu et al., 2017; Ugur et al., 2020). After supplementing the cells with exogenous biotin, biotin ligase results in the addition of biotin to lysine residues of proteins located in the proximity of the targeted locus (Fig 6). The cells are then lysed, and the chromatin-bound by the dCas9-BirA protein complex is fragmented.

The biotinylated proteins are then captured using streptavidin-coated beads, which have a high affinity for biotin. After capturing the biotinylated proteins, they are eluted and can be analyzed using mass spectrometry to identify the proteins that are associated with the targeted DNA sequence.

Fig 6: Addition of biotin to lysine residues of proteins located in the proximity of the targeted locus

How is enChIP different from traditional ChIP?

EnChIP and traditional ChIP both aim to identify proteins associated with a specific region of DNA, but the approaches differ in several key ways.

(i) Target specificity: In traditional ChIP, a specific antibody is used to capture the protein of interest, which is bound to the target DNA. The DNA is then fragmented, and the protein-DNA complexes are immunoprecipitated using the antibody against the protein of interest.

On the other hand, enChIP involves recruiting a catalytically inactive dCas9 protein to a specific region of DNA using a gRNA. The dCas9 is fused to tags such as FLAG, and the protein-DNA complex is crosslinked, fragmented, and immunoprecipitated using antibodies specific to the tag.

Because enChIP uses a gRNA to target a specific genomic region, it is generally more specific than traditional ChIP.

(ii) Detection and analysis: Traditional ChIP often uses PCR, microarrays, or next-generation sequencing to identify the regions of DNA that are bound by the protein of interest. Whereas in enChIP, the isolated protein-DNA complex are analyzed using mass spectrometry to identify the proteins associated with the target DNA sequence.

(iii) Sensitivity: enChIP is generally more sensitive than traditional ChIP because it can detect weaker protein-DNA interactions that may be missed by traditional ChIP.

Overall, enChIP offers several advantages over traditional ChIP in terms of target specificity and sensitivity, thus making it a valuable tool for studying protein-DNA interactions and identifying the proteins associated with specific genomic regions.

If you liked this article and want to know more about more about applications of CRISPR in gene editing and beyond, click the below links:

For book lovers:

For video lovers:

https://www.udemy.com/course/crispr-cas-system-applications-in-gene-editing-and-beyond/?referralCode=8ED7BBAEB7AE497D755F

References:

Fujita, T., & Fujii, H. (2013). Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Biochemical and biophysical research communications, 439(1), 132–136.

Schmidtmann, E., Anton, T., Rombaut, P., Herzog, F., & Leonhardt, H. (2016). Determination of local chromatin composition by CasID. Nucleus, 7(5), 476–484.

Liu, X., Zhang, Y., Chen, Y., Li, M., Zhou, F., Li, K., … & Xu, J. (2017). In situ capture of chromatin interactions by biotinylated dCas9. Cell, 170(5), 1028–1043.

Ugur, E., Bartoschek, M. D., & Leonhardt, H. (2020). Locus-specific chromatin proteome revealed by mass spectrometry-based CasID. The Nucleus, 109–121.

Fujita, T., Kitaura, F., Oji, A., Tanigawa, N., Yuno, M., Ikawa, M., … & Fujii, H. (2018). Transgenic mouse lines expressing the 3x FLAG‐dC as9 protein for enCh IP analysis. Genes to cells, 23(4), 318–325.

Hamidian, A., Vaapil, M., von Stedingk, K., Fujita, T., Persson, C. U., Eriksson, P., … & Mohlin, S. (2018). Promoter-associated proteins of EPAS1 identified by enChIP-MS–a putative role of HDX as a negative regulator. Biochemical and biophysical research communications, 499(2), 291–298.

Happy learning!

--

--