Psbbi bla

We utilized the pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid, obtained from Feng Zhang (Addgene #42230), as the basis for constructing CRISPR/Cas vectors. To generate the mAID donor plasmids, we modified constructs of the Kanemaki lab (Addgene #72827 and #121180). In order to incorporate mRuby2, we replaced mCherry2 in the donor plasmid (Addgene #121180).
For the rescue experiments, wild-type (WT) UHRF1 and each of the point mutants (M8R/F46V, Y188A, DAEA, G448D, and H741A) were cloned into pLenti6.2/V5-DEST (invitrogen). Likewise, WT DNMT1 and each of the point mutants (H170V, D381A/E382A/S392A, W464A/W465A, C1226W) were cloned into pSBbi-Bla (Addgene: #60526). To target DNMT3A and DNMT3B, we cloned the oligonucleotide sequences for gRNA into the lentiCRISPR v2-Blast vector (Addgene #83480). Additionally, we cloned the shRNA targeting TET2 into the pLKO.1-blast vector (Addgene #26655). Plasmids were generated using PCR, restriction enzymes, or Gibson Assembly Cloning techniques. All plasmids underwent sequencing prior to their utilization. The oligonucleotide sequences inserted into the lentiCRISPR v2-Blast vector and pLKO.1-blast vector are available in Supplementary Table 1.

We cloned WT DNMT1 and point mutant DNMT1 (F631A/F632A) to pSBbi-Bla (Addgene: 60526). All plasmids were sequenced prior to use. To establish stable expressing exogenous DNMT1 cell lines, we used the sleeping beauty system^{43 (link)}. Briefly, cells were grown in a six-well plate, then transposase vector (Addgene: 34879) and each pSBbi-Bla plasmids (EV, WT, F631A/F632A) were transfected using Lipofectamine 2000 (Thermo Fisher Scientific). Four days after transfection, cells were selected with 10 µg/mL Blasticidin for 1 week. To detect DNA methylation levels, LUMA and Pyrosequencing were done according to standard procedures. Depletion of the endogenous DNMT1 was confirmed by immunoblotting using rabbit anti-DNMT1 (#5032; CST, 1:1000 dilution), mouse anti-Tubulin (ab7291; Abcam, 1:4000 dilution), and rabbit anti-V5 (ab206566; Abcam, 1:100 dilution). F631A/F632A site-directed mutagenesis of DNMT1 was performed by inverse PCR using two primers containing the mutations (Forward: 5′- ATACTGCCGCCGCAGAGCAAATTGAAAAGGATG-3′, Reverse: 5′-TCTGCGGCGGCAGTATCGAAGATCTGGTAGACC-3′).

cDNA coding for wild-type human SCN4A C-terminally conjugated to mTurquise2 (mTq2), followed by the self-cleaving peptide P2A sequence to mediate ribosomal skipping and SCN1B (SCN4A-mTq2-P2A-SCN1B) was subcloned into expression vector pSBtet-Pur (addgene #60507) (Kowarz et al., 2015 (link)) by using the NEBuilder HiFi DNA Assembly Cloning Kit (NEB). The missense mutation E761K that blocks the canonical ionic pore (Schlief et al., 1996 (link)), was introduced to SCN4A using KOD-Plus mutagenesis kit (TOYOBO, Osaka, Japan). The resulting construct SCN4A^E761K (hNav1.4^pb) served as a control for gating pore current (Fig. 1A, gray). Hypokalemic periodic paralysis (HypoPP)-associated mutations R669H, R672H and R1135H were then introduced to hNav1.4^pb by using the KOD-Plus mutagenesis kit to generate R669H-hNav1.4^pb, R672H-hNav1.4^pb, R1135H-hNav1.4^pb and R669H/R672H (DMT)-hNav1.4^pb. Wild-type mouse Kir2.1 (mKir2.1) was a generous gift from Dr Yoshihiro Kubo. The cDNA coding for mKir2.1 was subcloned into pSBbi-Bla (Addgene #60526) using the NEBuilder HiFi DNA Assembly Cloning Kit (NEB).