Isolating Genomic Regions of Interest with the CRISPR System

While much of CRISPR research has focused on genome editing, numerous discoveries have been made using the Cas9 nuclease in the absence of genomic alterations. These studies utilize a catalytically inactive form of Cas9 known as dCas9 (Jinek et al., 2012). Like Cas9, dCas9 can bind to a specific DNA sequence via a targeting gRNA. But dCas9 does not cleave the DNA. Much of the research using dCas9 has focused on transcriptional activation using a fusion to a transcriptional activator such as VP64 (Gilbert et al., 2013), or repression of transcription through binding a promoter region to inhibit association of transcriptional activators (Qi et al., 2013). However, the fusion of dCas9 with a protein tag allows for the isolation of a genomic region of interest targeted by a gRNA.

CRISPR-mediated purification of a specific genomic region

In 2013, the Hodaka Fujii Laboratory first described a method to purify a specific genomic region using the CRISPR system, consisting of dCas9 and targeting gRNA, to identify molecular interactions at this site (Fujita and Fujii, 2013). They call this method engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) because any engineered DNA-binding molecule, such as Zinc-finger or TAL proteins, can be used. The Fujii lab demonstrated this in mammalian cells by expressing dCas9 with an N-terminal 3xFLAG tag (3xFLAG-dCas9) and a gRNA targeting their genomic locus of interest, the promoter region of the transcription factor IRF-1. Immunoprecipitation, using an antibody targeting the 3xFLAG tag, allowed these researchers to isolate dCas9 with the bound genomic locus, which they then confirmed by PCR. Subsequent mass spectroscopy studies allowed for the identification of proteins associated with the IRF-1 locus.

Figure 1: Schematic of enCHIP. Image from Fujita and Fuji, 2013.

Prior to the development of this method, isolation of a specific genomic region was an intensive process. One method required insertion of a recognition sequence into the genomic DNA that could then be recognized by a DNA-binding molecule (Hoshino and Fujii, 2009). Other forms of enChIP, that do not use dCas9, require Zinc-finger or TAL proteins targeting a specific genomic locus, which can require multiple cloning steps (Fujita et al., 2013). CRISPR-mediated purification of a specific genomic region alleviates these issues as a region of interest can be targeted using a gRNA, which can easily be cloned into a gRNA expression plasmid.

Experimental use

Since the initial report describing CRISPR-mediated purification of a specific genomic region, numerous adaptations of this system have been made in different fields of study. In addition to the identification of DNA binding proteins, this method can characterize nucleic acid-nucleic acid interactions at a genomic locus (Fujita et al., 2017). CRISPR-mediated purification of a specific genomic region has also recently been adapted to isolate genomic loci in bacteria (Fujita et al., 2018). Furthermore, the Fujii Lab generated a transgenic mouse line and retroviral expression system for using this approach in in vivo studies (Fujita et al., 2018).

In addition to the "in cell" form of this method, in which an engineered DNA-binding molecule such as the CRISPR complex is expressed in cells to be analyzed, the Fujii lab also developed another "in vitro" form. In this form, the CRISPR complex, consisting of a recombinant dCas9 protein and synthetic gRNA, is incubated with fragmented chromatin or library of purified DNA for purification of a specific chromatin or DNA fragment (Fujita et al., 2016). 3xFLAG-tag fused to dCas9, along with a biotinylated gRNA, were successfully used in this "in vitro" form.

Any tag system can be used to isolate genomic regions of interest with dCas9 and a targeting gRNA. One such example by the Xu lab is CRISPR affinity purification in situ of regulatory elements (CAPTURE) (Liu et al., 2017). This system utilizes dCas9 fused to a biotinylation tag expressed on a plasmid, allowing for in vivo biotinylation through a plasmid-based biotin ligase. Biotinylated dCas9, with bound DNA, can then be purified by streptavidin high-affinity purification. This system is both highly sensitive and specific and allowed the Xu lab to identify protein and nucleic acid binding partners at numerous genomic loci through proteomics and the chromosome conformation capture (3C) technique. In addition to 3xFLAG and biotin tags, use of tag systems including Protein A, 1xFLAG, 2xAM, and HA have been reported, as well as affinity purification with an anti-Cas9 antibody. These different tag systems and affinity purification schemes add additional flavors to this methodology.

Using this technique, researchers have begun to make important discoveries in various biological fields. In 2015, the Fujii Lab identified non-coding RNAs associated with telomeres by combining purification of a specific genomic region and RNA sequencing (Fujita et al., 2015). Using similar affinity purification with an anti-Cas9 antibody, the Li lab found a microRNA (miR483) that binds to the promoter of the growth factor IGF2 open reading frame to relax imprinting (Zhang et al., 2017). This upregulates expression of IGF2, a characteristic of many human malignancies. Additionally, the Tapscott Lab identified regulators of DUX4, a transcriptional regulator that when inappropriately expressed can cause facioscapulohumeral muscular dystrophy (FSHD), a disease for which there is currently no cure (Campbell et al., 2018).

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Conclusions

CRISPR-mediated purification of a specific genomic region is another example of the many tools based on targeting of a genomic locus through a specific gRNA-dCas9 complex. When combined with next-generation sequencing, this method allows for the recovery of a vast amount of data from a single experimental setup. As we’ve seen for many other CRISPR techniques, this method also has great potential for improvements and adaptations that can provide an increased understanding of DNA binding and gene regulation.

For additional information on CRISPR-mediated purification of a specific genomic region, other forms of enChIP, and additional research conducted in the Hodaka Fujii Laboratory, please also see our interview with Dr. Fujii.

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References

Campbell, Amy E., et al. "NuRD and CAF-1-mediated silencing of the D4Z4 array is modulated by DUX4-induced MBD3L proteins." Elife 7 (2018): e31023. PubMed PMID: 29533181. PubMed Central PMCID: PMC5849414.

Fujita, Toshitsugu, and Hodaka Fujii. "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 (2013): 132-136. PubMed PMID: 23942116.

Fujita, Toshitsugu, et al. "Identification of telomere-associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP)." Scientific reports 3 (2013): 3171. PubMed PMID: 24201379. PubMed Central PMCID: PMC3821016.

Fujita, Toshitsugu, et al. "Identification of non-coding RNAs associated with telomeres using a combination of enChIP and RNA sequencing." PLoS One 10.4 (2015): e0123387. PubMed PMID: 25874893. PubMed Central PMCID: PMC4395285.

Fujita, Toshitsugu, Miyuki Yuno, and Hodaka Fujii. "Efficient sequence‐specific isolation of DNA fragments and chromatin by in vitro enCh IP technology using recombinant CRISPR ribonucleoproteins." Genes to Cells 21.4 (2016): 370-377. PubMed PMID: 26848818.

Fujita, Toshitsugu, et al. "Identification of physical interactions between genomic regions by enCh IP‐Seq." Genes to Cells22.6 (2017): 506-520. PubMed PMID: 28474362.

Fujita, Toshitsugu, Miyuki Yuno, and Hodaka Fujii. "An enChIP system for the analysis of bacterial genome functions." BMC research notes 11.1 (2018): 387. PubMed PMID: 29898790. PubMed Central PMCID: PMC6001023.

Fujita, Toshitsugu, et al. "Transgenic mouse lines expressing the 3x FLAG‐dC as9 protein for enCh IP analysis." Genes to cells 23.4 (2018): 318-325. PubMed PMID: 29480524.

Gilbert, Luke A., et al. "CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes." Cell 154.2 (2013): 442-451. PubMed PMID: 23849981. PubMed Central PMCID: PMC3770145.

Hoshino, Akemi, and Hodaka Fujii. "Insertional chromatin immunoprecipitation: a method for isolating specific genomic regions." Journal of bioscience and bioengineering 108.5 (2009): 446-449. PubMed PMID: 19804873.

Jinek, Martin, et al. "A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity." Science337.6096 (2012): 816-821. PubMed PMID: 22745249. PubMed Central PMCID: PMC6286148.

Liu, Xin, et al. "In situ capture of chromatin interactions by biotinylated dCas9." Cell 170.5 (2017): 1028-1043. PubMed PMID: 28841410.

Qi, Lei S., et al. "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression." Cell 152.5 (2013): 1173-1183. PubMed PMID: 23452860. PubMed Central PMCID: PMC3664290. 

Zhang, Yiqun, et al. "CRISPR Cas9-guided chromatin immunoprecipitation identifies miR483 as an epigenetic modulator of IGF2 imprinting in tumors." Oncotarget 8.21 (2017): 34177. PubMed PMID: 27486969. PubMed Central PMCID: PMC5470959.

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