Background

Elucidation of molecular mechanisms underlining genome functions requires the identification of molecules that interact with the genomic region of interest in vivo. To this end, we have developed locus-specific chromatin immunoprecipitation (locus-specific ChIP) technologies consisting of insertional ChIP (iChIP) and engineered DNA-binding molecule-mediated ChIP (enChIP) (Fujita et al., 2016a). Locus-specific ChIP is a method to biochemically isolate a genomic region of interest from cells. Molecules interacting with that isolated genomic region are identified by biochemical analyses, such as mass spectrometry (MS) and next generation sequencing (NGS). In iChIP, an exogenous DNA-binding protein and its recognition DNA sequence are used for ‘in cell’ locus-tagging. In enChIP, engineered DNA-binding molecules, such as transcription activator-like (TAL) proteins and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), are used for ‘in cell’ locus-tagging. The tagged loci are isolated by affinity-purification. We recently developed in vitro enChIP, in which the locus-tagging was performed in vitro (in a test tube) using recombinant and/or synthetic engineered DNA-binding molecules (Figure 1) (Fujita and Fujii, 2014a and 2016b). Here, we describe a protocol of in vitro enChIP using CRISPR ribonucleoproteins (RNPs) followed by NGS (in vitro enChIP-Seq) for the identification of genomic regions that physically interact with the locus of interest (Fujita et al., 2017a).


Figure 1. Scheme of in vitro enChIP using CRISPR RNPs. Figure 1 reproduced under the Creative Commons Attribution License from: Fujita et al. Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins. Genes Cells. 2016; 21: 370-377.

Scheme of in vitro enChIP using CRISPR RNPs (Figure 1)
1. A nuclease-dead form of Cas9 (dCas9) fused to an epitope-tag(s) (e.g., 3xFLAG-dCas9) is prepared as a recombinant protein. Guide RNA (gRNA) that recognizes the DNA sequence of the genomic region of interest is generated chemically. As to gRNA, single gRNA (sgRNA) or a complex of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) can be used.
2. Cells to be analyzed are crosslinked, if necessary, and lysed, and DNA is fragmented.
3. The 3xFLAG-dCas9/gRNA complex is incubated with the fragmented chromatin DNA in a test tube. The genomic DNA bound to the 3xFLAG-dCas9/gRNA complex is affinity-purified using antibody against the epitope-tag(s) or dCas9 itself. Alternatively, the complex can be purified using tags fused to gRNA (e.g., biotin). The isolated complexes retain molecules that interact with the target locus.
4. Reverse crosslinking, if necessary, and subsequent purification of DNA, RNA, or proteins allows identification and characterization of these molecules. For example, NGS or MS can be combined to identify genomic regions or proteins that physically bind to the target locus.

We previously described a detailed protocol of ‘in cell’ enChIP for the identification of proteins that interact with a locus of interest in Bio-protocol (Fujita and Fujii, 2014b). Procedures for the preparation of sonicated chromatin in in vitro enChIP are the same as those for ‘in cell’ enChIP. In addition, other procedures (e.g., preparation of Dynabeads) are similar between them. Therefore, we recommend to refer to the ‘in cell’ enChIP protocol (Fujita and Fujii, 2014b) in parallel.