The nucleofection of DM1-iPSCs were carried out using Lonza P3 primary cell 4D nucleofection kit according to manufacturer’s protocol (Lonza). Prior to nucleofection, the DM1 –iPSCs were maintained in feeder free culture
condition as described previously. For the nucleofection reaction mix, a ribonucleoprotein (RNP) complex was prepared by mixing 60 pmol Cas9 protein (Integrated DNA Technologies) and 150 pmol of each sgRNA (5-CTGrepeatsgRNA, 3-CTGrepeat-sgRNA) (Synthego) followed by a 10
min incubation at room temperature (control conditions comprised the following: 60 pmol Cas9 and 300 pmol scrambled gRNA; 150 pmol 5-CTGrepeat-sgRNA, 150 pmol 3-CTGrepeat-sgRNA and no Cas9) mixes. Thereafter, DM1– iPSCs were dissociated into single cells with TrypLE express
(Thermo Scientific). Post dissociation, 5 × 105 DM1– iPSCs were resuspended in 20 l P3 nucleofection buffer and mixed with previously prepared RNP complexes and nucleofected using the ‘CA137’ program (using the Nucleofector 4D). Post-nucleofection, the cells were plated on geltrex-coated surface, in Essential 8™ medium (Thermo Scientific) with 10M rock inhibitor (Y-27632, Stem cell Technologies). A day post-nucleofection, cells were supplemented
with fresh Essential 8™ medium (Thermo Scientific) and cultured for further downstream experiments. DM1– iPSCs treated with Cas9 protein and sgRNA (5-CTGrepeatsgRNA, 3-CTGrepeat-sgRNA) were cloned by limiting dilution.
Dissociated DM1–iPSCs were plated as single cells on vitronectin (Stemcell Technologies) coated 96-well plate with Clone R supplement and mTeSR culturing medium (Stemcell Technologies). After 10–15 days of culturing with regular media change, single clones were established. These
DM1-iPSC clones obtained were transferred to larger surface area and maintained as iPSCs clonal lines until further analysis.