PacI (10 U/µL)

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	To replace VP64 with the catalytic domains, we used sticky-end ligation after digestion with fast-digest restriction enzymes MluI and PacI (Thermo Scientific).

Protocol tips
To replace VP64 with the catalytic domains, we used sticky-end ligation after digestion with fast-digest restriction enzymes MluI and PacI (Thermo Scientific).

Publication protocol

pMLM3705 and MLM3636 plasmids were a gift from Keith Joung (Addgene plasmid #47754 and #43860, respectively). An additional multiple cloning site was added by replacing the VP64 activator in dCas9-VP64 with a sequence containing a PacI restriction site (new plasmid referred to as dCas9-Empty). The catalytic domain of human histone methyltransferase PRDM9 was amplified from total complementary DNA (cDNA) of a testicular cancer cell line, and the ubiquitin-conjugating enzyme UBE2A and histone methyltransferase DOT1L catalytic domains from human fibroblasts by Pfu DNA polymerase (Thermo Scientific, Leon-Rot, Germany), using forward and reverse primers introducing MluI and PacI restriction sites at the 5′ and 3′ ends, respectively (Supplementary Table 1; Supplementary Note 1). These catalytic domains were introduced into dCas9-Empty using sticky-end ligation after digestion with AscI and PacI digestion with T4 ligase (Thermo Scientific). Cloning of gRNAs was achieved as previously described19. Briefly, pairs of DNA oligonucleotides encoding 20 nt gRNA targeting sequences were annealed together to create double-stranded DNA fragments with 4 bp overhangs (Supplementary Table 2). These fragments were ligated into BsmBI-digested plasmid pMLM3636. Irrelevant gRNAs were designed to bind regions of the mouse genome. EpCAM targeting ZF protein (ZFA and ZFB)28, ICAM targeting ZF protein (ZFC, kindly provided by C.F. Barbas III, the Scripps Institute, La Jolla, CA, USA)25, RASSF1a targeting ZF proteins (ZFX and ZFZ; self-engineered) and PLOD2 targeting six ZF proteins (ZF2 and ZF8)62 were used for epigenetic editing of the respective gene promoter (Supplementary Table 3). To replace VP64 with the catalytic domains, we used sticky-end ligation after digestion with fast-digest restriction enzymes MluI and PacI (Thermo Scientific). Each ZF effector domain construct contains a nuclear localization signal and a terminal haemagglutinin (HA) decapeptide tag. We verified all PCR-cloned constructs by DNA Sanger sequencing (Baseclear, Leiden, The Netherlands). The enzymatically inactive pMX-ZFA-MutPRDM9 mutant (G278 to A278) was obtained by site-directed mutagenesis on wild-type pMX-ZFA-PRDM9. The enzymatically inactive dCas9-MutDOT1L mutant (NN241–242 to AD241–242) was obtained by site-directed mutagenesis on wild-type dCas9-DOT1L.

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