In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

The epigenetic silencing technology that we developed belongs to a larger arena of technologies, which are gene silencing technologies. Before we started to develop epigenetic silencing, there were two main alternatives in the field. One was gene knock-down by RNA interference, and the other was gene disruption by artificial nucleases.

Gene disruption by artificial nucleases works at the DNA level. So you have your target gene that you want to inactivate, and you deliver to the cell some artificial nucleases, which induce a double-strand DNA break. This kind of double-strand breaks can induce frameshift mutations that finally impose either the generation of a non-functional protein or, simultaneously, the transcript’s degradation.

On the other hand, epigenetic silencing works by delivering into the cell the so-called Engineered Transcriptional Repressors. These are some small proteins that are able to bind to your target DNA, and then impose on your target gene the so-called epigenetic marks. These are some chemical modifications that, once deposited on the DNA, can lead to chromatin compaction.

Chromatin compaction importantly then leads to transcriptional inhibition, so you don't have any more expression of your target gene. And very importantly, this kind of transcriptional repressive state can be prolongedly inherited by the cells. To identify cell lines that express the target gene to be silenced, browse the target gene in The Human Protein Atlas and navigate through the Cell Lines section to identify cell lines representative of the somatic tissue of interest.

From the identified cell lines, prioritize the ones for which efficient transient gene delivery protocols are available. Next, open a gene viewer and identify the region of the target gene to integrate the fluorophore expression cassette. Then, use CHOPCHOP to identify and select gRNAs that will cut in the target region. In this case, the first intron of the B2M gene.

Next, design a donor template for the gRNAs cut site, consisting of a left homology arm, a promoter-free transgene expression cassette, and a right homology arm. Deliver the CRISPR Cas9 nuclease system and the donor template inside the K-562 cell line through nucleofection, according to the manufacturer's instructions. Culture the K-562 cells for at least 14 days.

Monitor the expression levels of the fluorescent reporter over time by using a flow cytometer. Activate the phycoerythrin channel to measure the fluorescence intensity of the tdTomato reporter. Browse the target gene in the UCSC Genome Browser and extract the nucleotide sequence of regions that potentially regulate its transcriptional activity, such as CpG islands and sites enriched for H3K27 acetylation.

Paste the selected sequences in the CHOPCHOP online tool, and select repression as the purpose of the gRNAs to be retrieved. CHOPCHOP will provide a list of gRNAs mapped on the genetic sequence of interest and listed according to a score, considering the number of off-target matches and the predicted on-target efficiency. Select at least 10 gRNAs per target sequence.

Try to select gRNAs spanning the whole region to be targeted with no full matches with other intergenic sequences throughout the genome. After transforming the plasmids encoding for the ETRS in chemically competent E.coli cells, screen the colonies for the presence of the ETRs-encoding plasmids by restriction enzyme digestion and Sanger sequencing. To clone the gRNAs inside the phU6 gRNAs backbone, first, generate oligos using a molecular biology design software.

Append a five nucleotide sequence upstream of the protospacer and label this 25 nucleotides-long oligo. Similarly, append a five nucleotide sequence to the reverse complement of the protospacer and a cytosine downstream of it to generate a 25 nucleotide-long oligo and label it. Get these oligos synthesized as salt-free single-stranded DNA oligos resuspended in water at a 100 micromolar concentration.

Add one microliter of each oligo to two microliters of annealing buffer and 16 microliters of water. Perform oligo annealing by placing the solution in a thermocycler at 95 degrees Celsius for 10 minutes. Then, gradually cool to 25 degrees Celsius over 45 minutes.

Dilute one microliter of the annealed oligos with 99 microliters of nuclease-free water. Then, ligate one microliter of this dilution with 50 nanograms of phU6 gRNAs plasmid, previously digested with the Bsa1 restriction enzyme. Transform 20 microliters of chemically competent E.coli cells with two microliters of the ligation product.

Pick multiple colonies for plasmid DNA production and confirm successful cloning of the protospacer by Sanger sequencing. To deliver the gRNAs and CRISPR/dCas9-based ETRs in the reporter cell line in an array, first, prepare separate tubes containing 500 nanograms of each of the plasmids and add 125 nanograms of different gRNAs encoding-plasmids to be tested. Include a gRNAs- and ETRs-free nucleofection condition as a mock-treated sample.

Prepare at least three technical replicates per sample. Pellet 5 x 10⁵ B2M tdTomato K-562 cells per tube and nucleofect them with the plasmid mix. Resuspend the cells in 200 microliters of previously warmed RPMI 1640 mammalian cell culture media and place them in the incubator.

Use flow cytometry to measure the percentage of silenced cells at different time points after delivery of the ETRs. Use wild type cells without the tdTomato-encoding sequence to set the threshold of reporter-negative cells.

Use the mock-treated sample to set the gate for reporter-positive cells. Identify the top three gRNAs in terms of long-term silencing efficiency. Use FACS to select the reporter-negative subpopulation stably maintained in those samples.

Also, perform FACS of the mock-treated samples under the same treatment to allow proper comparison in subsequent analyses. A tdTomato fluorescent reporter was integrated in the first intron of the human B2M gene by CRISPR/Cas9-induced homology-directed repair. Reporter-positive K-562 cells appear in the treated sample after integration.

Upon transient delivery of the triple ETRs combination, together with gRNAs, longitudinal flow cytometry analysis was performed to assess the expression of the fluorescent reporter. A peak was observed in reporter repression at acute analyses, which was partially reabsorbed due to mitotic dilution of the ETRs-encoding plasmids over time. Effective deposition of CpG methylation on the target locus by the ETRs and gRNAs combination was evident by permanent repression of the reporter in a sizable fraction of treated cells. Different gRNAs showed different long-term silencing efficiency.

One of the key steps to achieve permanent and efficient epigenetic silencing is the identification of effective gRNAs. To do so, for any kind of application, you need to start with the identification of the most responsive sequences, including promoters and enhancers in any other sites for transcriptional regulation.

Once the most responsive and effective region is identified, one should design different gRNAs and test them individually or in combination. One of the most obvious applications of epigenome editing is diseases caused by gain-of-function mutations. Alternatively, we should anyway keep in mind that the ability to specifically change the epigenetic regulation of a given promoter could represent a super useful tool for both translational and basic science.