Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli

Targeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Although the technique has high potential for studying the role of DNA methylation in higher eukaryotes, its usefulness is hampered by insufficient methylation specificity. One of the approaches proposed to suppress methylation at unwanted sites is to use MTase variants with reduced DNA binding affinity. In this work we investigated how methylation specificity of chimeric MTases containing variants of the CG-specific prokaryotic MTase M.SssI fused to zinc finger or dCas9 targeting domains is influenced by mutations affecting catalytic activity and/or DNA binding affinity of the MTase domain. Specificity of targeted DNA methylation was assayed in E. coli harboring a plasmid with the target site. Digestions of the isolated plasmids with methylation sensitive restriction enzymes revealed that specificity of targeted DNA methylation was dependent on the activity but not on the DNA binding affinity of the MTase. These results have implications for the design of strategies of targeted DNA methylation.

. Nucleotide sequence of the original target regions containing a Ppu21I and/or an AvaI site. Yellow and green highlighting indicate the 6ZA and 6ZB zinc finger binding sites, respectively. To indicate positions of restriction sites used for plasmid construction, partial sequences preceding the 6ZA and following the 6ZB binding site are shown for the T0 target region. The CG sites located between the 6ZA and 6ZB binding sites are highlighted by grey background. The blue numbers indicate the distances in base pairs between the 6ZB binding site and the closest CG in the target region.

Plasmids encoding 6ZB-M.SssI fusions
The plasmid pBHH-MSssI, a derivative of pBNH-MSssI 2 , contains the gene of M.SssI MTase cloned in the expression plasmid vector pBAD24 3 . It was constructed by replacing the SacI-PstI fragment of pBNH-MSssI with the SacI-PstI fragment of pBHNS-MSssI 4 . The fragment replacement added a Cterminal His6-tag to the M.SssI variant encoded by the plasmid. To construct N-terminal fusion between M.SssI and the zinc finger protein 6ZB, the 6ZB coding sequence was PCR-amplified using pcDNA3.1mnhk up2 plasmid DNA as template and oligonucleotides AK231 and AK232 as primers. The PCR product was T/A-cloned in pTZ57R/T (Thermo Scientific), then the 6ZB coding sequence was excised by NcoI-XhoI double-digestion and inserted between the NcoI and XhoI sites of pBHH-MSssI. Replacing the original NcoI-XhoI fragment of pBHH-MSssI with the PCR product deleted the N-terminal His-tag of the MTase and created in-frame fusion between the 6ZB and M.SssI genes. This plasmid was named pB6ZB-MSssI (Supplementary Figure S2). Variants of pB6ZB-MSssI expressing fusion proteins with mutant MTase domain (C141S, Q147L or T313H) were constructed by fragment replacement using the respective mutant M.SssI allele described previously 4,5 .
Supplementary Figure S2. Map of pB6ZB-MSssI with restriction sites used for plasmid construction.
To increase the distance between the 6ZB binding site and the Ppu21I site in pZB-MSssI(T313H)-T0, 4 base pairs were inserted by cutting the plasmid at the unique Cfr9I site (Supplementary Figure S1), and filling-in the ends by Klenow polymerase to yield the plasmid pZB-MSssI(T313H)-T0+4 ( Figure 2). Ligation of the filled-in Cfr9I ends created a unique Eco52I site (CGGCCG). Additional 4 base pairs were inserted by cutting pZB-MSssI(T313H)-T0+4 with Eco52I, filling-in the ends and circularization of the molecule. The new plasmid was named pZB-MSssI(T313H)-T0+8 (Supplementary Figure S1). To construct a series of plasmids with varying distances between the 6ZB binding site and the addressed AvaI site, the SalI-SphI fragment carrying the original T0 target region and the flanking ZF binding sites (Supplementary Figure S1) was transferred from pZB-MSssI-T0 into the plasmid vector pOK12 6 . The goal of this cloning step was to obtain a plasmid (pOK-ZABT), in which the EcoRI and Kpn2I sites bordering the target region were unique. The original T0 target region in pOK-ZABT was replaced with new target regions by cloning the double-stranded oligonucleotides AK284-285, AK286-287, AK288-289, AK290-291 or AK296-297 (Supplementary Table S2) between the EcoRI and Kpn2I sites of the plasmid. The new target regions together with the flanking zinc finger binding sites were subsequently re-inserted, on SalI-SphI fragments, into the pZB-MSssI(T313H) backbone. In the name of the resulting plasmids the target region is indicated by an extension referring to the ˮtop strandˮ of the inserted duplex, e.g. pZB-MSssI(T313H)-T284, pZB-MSssI(T313H)-T286, etc. (Supplementary Table S1, Supplementary Figure S1). The last member of this series, pZB-MSssI(T313H)-T296, contains a unique Acc65I site (GGTACC) overlapping the AvaI site (Supplementary Figure S1). The distance between the 6ZB binding site and the AvaI site was further increased by filling in the Acc65I site of pZB-MSssI(T313H)-T296 to obtain pZB-MSssI(T313H)-T296-20, or by inserting the self-complementary oligonucleotides AK215 or AK219 into the Acc65I site to yield pZB-MSssI(T313H)-T296-215 and pZB-MSssI(T313H)-T296-219, respectively (Supplementary Figure S1).
To increase the distance between the 6ZB zinc finger domain and the MTase, the double-stranded oligonucleotide AK280-AK281 (Supplementary Table S2) was inserted into the unique XhoI sites of some pZB-MSssI(T313H) and pZB-MSssI(C141S) plasmids. The AK280-281 duplex was designed to restore, upon insertion, only one of the two bordering XhoI sites, thus allowing sequential addition of multiple copes of the duplex. Each insertion step added a copy of the LEGGGSG peptide to the interdomain linker. The number of linker insertions were indicated by the extensions L1, L2, L3, L4 or L5 in the name of the plasmid ( Figure 2C, Supplementary Figure S4).

Plasmids encoding M.SssI-6ZA fusions
The scheme of construction is shown on Supplementary Figure S5. The plasmid pBS-CAL75 carries the 3'-half (peptide [242-386]) of a modified version of the M.SssI gene, in which a HpaI site overlaps the stop codon 2 . A derivative of this plasmid (pBS-CAL7578) encoding the T313H substitution was constructed by site-directed mutagenesis using the mutagenic oligonucleotide AK159. The gene of the 6ZA zinc finger protein in pcDNA3.1mnhk up1 contains a HindIII site, which would have interfered with a planned cloning step. This HindIII site was eliminated by site-directed mutagenesis leaving the amino acid sequence intact. The modified 6ZA gene lacking the HindIII site was PCR-amplified using the primers AK259 and AK260. To facilitate fusion construction between the M.SssI(T313H) and 6ZA genes, AK259 and AK260 contained a PvuII and a PstI site, respectively as 5'-extension (Supplementary Table  S2). The PCR product was cloned in pTZ57R/T (Thermo Scientific) to obtain pTZ-6ZA. The fragment containing the 6ZA coding sequence was excised from pTZ-6ZA by PvuII and PstI double digestion and cloned between the HpaI and PstI sites of pBS-CAL7578 (Supplementary Figure S5). Ligation of the HpaI end to the PvuII end created in-frame fusion between the M.SssI(T313H) and the 6ZA gene.  Figure S5). The T286+4 target region (Figure 1) was added to this plasmid by inserting the 748 bp PstI fragment of pZB-MSssI(T313H)-T286+4 into the unique PstI site downstream of the stop codon of the M.SssI-6ZA gene. The new plasmid was named pMSssI(T313H)-6ZA-T286+4. The related plasmids pMSssI(wt)-6ZA-T286+4, pMSssI(C141S)-6ZA-T286+4 and pMSssI(Q147L)-6ZA-T286+4 encoding the wild-type, the Q147L or the C141S variant of M.SssI Cterminally fused to the 6ZA domain were constructed from pMSssI(T313H)-6ZA-T286+4 by fragment replacement using plasmids described in 4,5 .
The plasmid pOK-CRISPR-t (Kn R ) was used to express the guide RNA for CRISPR-mediated targeting. It was constructed by first cloning the 271 bp NcoI-EcoRI fragment fragment of pCRISPR 8 containing one copy of the CRISPR array between the NcoI and EcoRI sites of pOK12 6 , then by inserting the PCRamplified tracrRNA gene into the HindIII site of the plasmid. The tracrRNA gene was synthesized using pdCas9 7 as template and AK344 and AK345 as primers. The plasmid pOK-CRISPR-t-735 contains the AK735-AK736 duplex cloned between the BsaI sites of pOK-CRISPR-t. The dCas9 binding site determined by pOK-CRISPR-t-735 overlaps the 6ZA zinc finger binding site (Figure 1).

Effect of hemimethylation on Ppu21I, AvaI and Bsh1285I cleavage
For Ppu21I a 1267 bp fragment containing two Ppu21I sites was synthesized using pZB-MSssI(C141S)-T0 as template and AK431, AK432 and AK433 as primers (Supplementary Table S2). When AK432 was used as forward primer, one of the Ppu21I sites was hemimethylated (Supplementary Figure S6).
For AvaI, a PCR template was created by cloning the 645 bp BamHI fragment of pB6ZB (see above) into pBluescript II SK+ to yield pBS-6ZB. The purpose of cloning the BamHI fragment was to introduce additional AvaI sites in the PCR fragment to be synthesized. PCR fragments were synthesized using pBS-6ZB as template, AK322 (unmethylated) or AK368 (methylated) as forward primers and AK361 as reverse primer (Supplementary Table S2). The primers defined a 752 bp PCR product containing three AvaI sites (Supplementary Figure S7). Figure S6. Sensitivity of Ppu21I to hemimethylation of the substrate site. (a), Scheme of the 1267 bp PCR fragment synthesized using the AK431, AK432 and AK433 primers. The forward primers contained a Pvu21I site (TACGTA), and differed in the methylation status of the site. Vertical arrows, PpuI sites; horizontal arrows, PCR primers. CG sites are underlined. Methylation state of the Ppu21I site in the primer is shown below the arrow representing the primer: C5-methylcytosine is in red font. Numbers above the horizontal bar indicate the length of fragments generated by Ppu21I digestion. For Bsh1285I the plasmid pBS-6ZB was used as template, the oligonucleotides AK378/379/380/381 as forward primers and AK382 as reverse primer (Supplementary Table S2). The 840 bp PCR fragment contained three Bsh1285I sites ( Figure 5).

Supplementary
The PCR fragments were purified using GeneJET PCR Purification Kit (Thermo Scientific), and digested with the restriction enzyme of interest. The digestion products were analyzed by agarose gel electrophoresis.
Supplementary Figure S8. Targeted DNA methylation in E. coli by 6ZB-M.SssI variants that differ in the M.SssI domain, in the interdomain linker, and/or in the target region. Cultures of E. coli ER1821 harboring variants of pZB-MSssI were induced with arabinose for 6ZB-M.SssI expression. Plasmids prepared from the cultures were digested with Bsh1285I. The blue numbers under the gel show the distance in base pairs between the 6ZB binding site and the first CG of the Bsh1285I site in the target region ( Figure 1). The number of linker peptides (L1 through L5) inserted into the interdomain region is indicated below the gel on panel (b). The two parental fragments and the resulting protected fragment are marked with white circle and white asterisk, respectively. Quantitative evaluation of restriction protection at the targeted Bsh1285I site is shown on the right. The relative molar amounts of the parental and the resulting protected fragments calculated using the program ImageJ are shown on the bar diagram: empty, small parental fragment; dotted, large parental fragment; striped, protected fragment. Figure S9. Targeted DNA methylation in E. coli by the C141S and T313H M.SssI variants N-terminally fused to the dCas9 targeting domain. Cultures of E. coli ER1821 harboring pB-dCas9-L1-MSssI(C141S)-T0+4 or pB-dCas9-L1-MSssI(T313H)-T0+4 plus pOK-CRISPR-t-735 were induced with arabinose for dCas9-L1-M.SssI(C141S or T313H) expression. Plasmids were prepared after different lengths of induction as indicated (in minutes) above the lanes, and were digested with Bsh1285I. For a map of the plasmids with Bsh1285I sites see Figure 4. The two parental fragments and the resulting protected fragment are indicated by white circle and asterisk, respectively. M, GeneRuler 1 kb DNA ladder, Thermo Scientific. Figure S10. Images of uncropped gels (Figures 2, 3, 4

and 5), see below Supplementary
Tables S1 and S2.       Evaluation of Figure 4b Supplementary Figure S12. Targeted DNA methylation in E. coli by 6ZB-M.SssI variants (biological repeat of the experiment shown on Figure 2). Cultures of E. coli ER1821 harboring pZB-MSssI-T0+4 (wild-type or mutant) were induced with arabinose for 6ZB-M.SssI expression. Plasmids prepared from the cultures were digested with Bsh1285I. Time course of plasmid methylation. Plasmids were prepared after different lengths of induction as indicated above the lanes. The two parental fragments and the resulting protected fragment are indicated by white circle and white asterisk, respectively. M, GeneRuler 1 kb DNA ladder, Thermo Scientific.