CRISPR-On System to Activate Endogenous Human Insulin (INS) Gene

Supplementary Material

CRISPR-on system to activate endogenous human insulin (INS) gene

Gimenez CA et al.

Supplementary Table 1. Oligonucleotides used for cloning on sgRNAs expression plasmid (pSPgRNA). Underlined letters indicate overhangs compatible to the BbsI- digested vectors. Bold letters indicate mismatch in target sequence (first G allows efficient U6 transcription from expression plasmids).

Target / Name / Forward Oligo 5´-3´ / Reverse Oligo 5´- 3´ / Reference
Oct-4 (mouse) / T1 / CACCGAGACGTCCCCAACCTCCGTC / AAACGACGGAGGTTGGGGACGTCTC / Hu et al
T2 / CACCGAACCTCCGTCTGGAAGACAC / AAACGTGTCTTCCAGACGGAGGTTC
NT3 / CACCGTGTGGGGGTGGGAGAAACTG / AAACCAGTTTCTCCCACCCCCACAC
INS (human) / sgRNA1 / CACCGGGGCTGAGGCTGCAATTTC / AAACGAAATTGCAGCCTCAGCCCC / n/a
sgRNA2 / CACCGCCAGCACCAGGGAAATGGTC / AAACGACCATTTCCCTGGTGCTGGC
sgRNA3 / CACCGCTAATGACCCGCTGGTCCTG / AAACCAGGACCAGCGGGTCATTAGC
sgRNA4 / CACCGAGGTCTGGCCACCGGGCCCC / AAACGGGGCCCGGTGGCCAGACCTC

Supplementary Table 2. Primers used for RT-PCR, quantitative PCR (qPCR) , DNA and RNA methylation analysis (PCRbis) , ChiP-qPCR and ChIP-BA analysis.

Forward primer (5’ to 3’) / Reverse primer (5’ to 3’)
RT-PCR
INS / GCAGCCTTTGTGAACCAACACC / TGTTCCACAATGCCACGCTTC
ACTB / CCTGGCACCCAGCACAAT / GCCGATCCACACGGAGTACT
qPCR
INS / ATCAGAAGAGGCCATCAAGCA / TAGAGAGCTTCCACCAGGTGTGA
ACTB / CCTGGCACCCAGCACAAT / GCCGATCCACACGGAGTACT
PCR bis
INS (DNA)
INS (RNA) / AATTTAGTTGAGAGAGAAAATTGGA
GGTTATTAAGTAGATTATTGTTTTTTTGTT / CCATATACAAACACRCAAAAA
TTAAGTAGATTATTGTTTTTTTGTTATGGT
ChiP-qPCR
INS / GGGCCCCTGGTTAAGACTCT / TGCAATTTCCGGACCATTTC
ChIP-BA
INS1 / TTAGTTGTGAGTAGGGATAGGTTTG / CATTTCCCTAATACTAAATCTATAAAAAAA
INS2 / TAGATTTAGTATTAGGGAAATGGTT / AAAAACTAAAAACTACTAAACCCCC

Supplementary Figure 1. CASP3 expression in the long term experiment showed an increase up to 2,5 fold at 30d post-transfection (HEK293T cells with all sgRNAs) by RT-qPCR. Technical duplicates were averaged for each gruop. Control groups cells were transfected with the dCas9-VP160 plasmid and empty sgRNA expression plasmids.

Supplementary Figure 2. Fold change values of mRNA expression in control human pancreas respect to HEK293T cells co-transfected with dCas9-VP160 and multiple sgRNAs, as measured by qPCR. The gene expression levels were normalized to the β-actin. The data is shown as the mean ± the SD (n= 2 biological replicates).

Supplementary Figure 3. (a) RNA methylation analysis of a region of the INS mRNA in human pancreas control and HEK293T CRISPR-on for two biological replicates (Rep 1 and Rep 2). All sgRNAs were used on CRISPR-on groups. The analyzed region ranges from the nucleotide number 60 to number 172, regarding the mRNA initial nucleotide, and contains 63 cytosines totally. Only cytosines that showed detectable level of methylation are shown. (b) Bisulfite non-converted INS mRNA sequences and electropherograms from INS bisulfite converted mRNA from the HEK293T CRISPR-on cells (Rep1) and human pancreas cells. The sequence ranges from nucleotide 56 to 165 regarding the initial nucleotide of human INS mRNA. Red boxes indicate cytosines showing detectable level of methylation.

Supplementary Figure 4. Location of the sgRNAs in human INS gene promoter regarding CG dinucleotides subjected to methylation analysis (- 250 to TSS). Arrows enclose each sgRNA target sequence. Squares indicate the PAM, a triplet NGG site immediately downstream of target. CRISPR-on system recognized this triplet, which is absolutely necessary for the hybridization of sgRNA toDNA target and gene activation. The sgRNAs can join the plus or minus chains. "N"symbolizes any nucleotide.

Supplemental Experimental Procedures

RNA methylation analysis

RNA purified from CRISPR-on HEK293T cells and control human pancreas was trated according to Schaefer and colleagues with minor modifications (Schaefer M., 2015). cDNA was obtained by RT using random hexamers. Primers used are described in Supplementary Table 1. The PCR products were purified and directly sequenced (Macrogen, Japan) and the methylation levels were measured as described by Jiang and colleagues (Jiang et al., 2010).

Supplemental References

Hu J, Lei Y, Wong WK, Liu S, Lee KC, He X et al. Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors. Nucleic Acids Res 2014; 7:4375-90.

Jiang M, Zhang Y, Fei J, Chang X, Fan W, Qian X et al. Rapid quantification of DNA methylation by measuring relative peak heights in direct bisulfite-PCR sequencing traces. Lab Invest 2010; 90(2):282-90.

Schaefer M. RNA 5-Methylcytosine Analysis by Bisulfite Sequencing. Methods Enzymol.2015;560:297-329.