Plasmid Construction

The expression plasmids for Cas9 nuclease (pSpCas9(BB)-2A-GFP) and Cas9 nickase, a D10A mutant version of Cas9 nuclease (pSpCas9n(BB)-2A-GFP), were gifts from Dr. Feng Zhang (Addgene, plasmid nos. 48138 [http://addgene.org/48138] and 48140 [http://addgene.org/48140]). The target sites of sgRNA were selected using the online CRISPR design tool (http://zlab.bio/guide-design-resources). Based on the input of the sequences flanking the 5′ and 3′ regions of the CTG repeat of the DMPK gene, several targeting sites were chosen to generate the fewest number of off-target sites as close to the CTG repeat as possible. The sgRNAs were cloned into the plasmids as described previously.26 Briefly, the top and bottom strands of oligonucleotides with BbsI restriction sites on their 5′ termini were chemically synthesized at FASMAC (Atsugi, Japan). The oligonucleotides were phosphorylated and annealed in a thermal cycler using the following program: 37°C for 30 min; 95°C for 5 min; ramp down to 25°C at 5°C/min. Then, pSpCas9(BB)-2A-GFP and pSpCas9n(BB)-2A-GFP were digested by BbsI (NEB, Ipswich, MA, USA) and ligated with each sgRNA. The insertions of the sgRNAs were confirmed by Sanger sequencing. For the CRISPRi experiments, AAV CMV-dSaCas9-KRAB-bGHpA, gifted by Dr. Charles Gersbach (Addgene, plasmid no. 106219 [http://addgene.org/106219]) and EF1-RFP-U6-gRNA (System Biosciences, Palo Alto, CA, USA) were used to express dCas9 and sgRNA, respectively.72 The TSS of the human DMPK gene was identified using the online database FANTOM5 (https://fantom.gsc.riken.jp/5/).73 sgRNAs with a PAM sequence of NNGRRT or NNGRR were designed in the vicinity using Benchling software (https://www.benchling.com). The corresponding oligonucleotides were synthesized, annealed, and ligated to the EF1-RFP-U6-gRNA plasmid according to the manufacturer’s instructions. The sgRNA sequences used are listed in Table S3.