*2.8. Chromatin Immunoprecipitation Assay (ChIP)*

ChIPs were performed as described previously [43] with the following modifications. Briefly, 2 <sup>×</sup> 107 cells were crosslinked using 1% formaldehyde for 7 min, the reaction was quenched with 125 mM glycine and cells were collected by scraping. After lysis, nuclei were resuspended in Sonication buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate, protease Inhibitor cocktail) and sonicated (Bioruptor, Diagenode) to obtain fragments of approximately 150–300 bp, verified on agarose gel electrophoresis. Samples were centrifuged at 13,000 rpm for 10 min at 4 ◦C and supernatants recovered and quantified by Bradford assay. One hundred micrograms of chromatin were immunoprecipitated with 5 μg of anti-NF-YB (GeneSpin) and anti-FLAG (Sigma-Aldrich) antibodies. Protein-G beads (KPL) were used for recovery of antibody-bound proteins. Crosslinking was reversed by incubation at 65 ◦C overnight. Reactions were digested with RNase A and Proteinase K and DNA purified using the DNA purification kit (PCR clean Up, GeneSpin). The DNA was eluted in TE (10 mM Tris-HCl pH 8, 1 mM EDTA) and used in real-time PCR. Three independent biological replicates of ChIP experiments were performed.

#### **3. Results**

### *3.1. Ablation of NF-YA Exon 3 in C2C12 Cells by a Four Guides CRISPR*/*Cas9n Strategy*

Mouse C2C12 cells mostly express NF-YAl [35–38]. To study the role of this isoform in maintenance and differentiation of C2C12, we set out a strategy to selectively eliminate exon 3, coding for the 28 extra amino acids present in NF-YAl and absent in NF-YAs. We figured that the use of four guides flanking precisely the exon 3 regions and of the single strand-cutting Cas9-nickase (Cas9n) would minimize off-target effects, which potentially affect the outcome of this technology [44]. Figure 1A shows the design of the four guide oligonucleotides, two couples targeting the 5' and 3' intronic DNA flaking exon 3, respectively. The two couples of oligos were first checked for absence of common genomic targets (Table S1) and cloned unpaired in the final pX330A\_D10A-1x2\_ad and pX330A\_D10A-1x2\_cb (Figure S1A), also expressing the Cas9n gene. The two plasmids were transfected in growing C2C12 cells by electroporation. Individual clones were isolated, expanded and analyzed by PCR, employing the amplicons shown in Figure 1B. As expected, the strategy was less efficient if compared to the standard use of two guides plus wt Cas9: 335 clones were individually screened and two were positive for correct ablation in homozygosity, as shown in Figure 1C. The results of PCRs of the two positive clones, #83 and #117, show the expected bands for the A, B and C amplicons, absent in the DNA of the parental C2C12 cells. The regions surrounding exon 3 in both clones were then amplified and sequenced: Figure S1B confirms the deletion of coding sequences of exon 3, with somewhat different ends in the two clones. In summary, we successfully ablated NF-YA exon 3, deriving two clones termed C2-YAl-KO. To the best of our knowledge, this is the second system of genome editing describing a clean deletion of an individual exon [45] and the first one employing the Cas9 nickase system coupled with four gRNAs.

**Figure 1.** Strategy for ablation of NF-YA exon 3 in C2C12 cells using CRISPR/Cas9n and four gRNAs. (**A**) Gene editing strategy for NF-YA exon 3 deletion using the Cas9-nickase (Cas9n) and four guide RNAs. The targeted sequence by each guide RNA and the deletion sites are shown. Note that Cas9n cuts only the DNA strand that is complementary to and recognized by the gRNA, making necessary the simultaneous presence of two gRNAs/Cas9n complexes to induce a double-strand break (DSB). (**B**) The three primer pairs used to check for positive C2-YAl-KO clones are shown with the specific amplification products highlighted by the dashed lines. (**C**) Example of PCR products run into a 1.2% agarose gel. The expected bands in control cells (ctr) are marked with arrowheads; clones #83 and #117 represent positive C2-YAl-KO clones.
