CRISPR sgRNA Design and Synthesis

The online tool CCTop (Stemmer et al., 2015 (link)) was used to design sgRNAs. sgRNAs were generated as previously described by Hwang et al., 2013 (link). Oligonucleotides were annealed and ligated into DR274 vector (Addgene), which was previously linearized using BsaI-HF (New England Biolabs). Template for in vivo transcription of sgRNA was amplified by PCR. In vivo transcription was performed using the HiScribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs) and RNA was purified using the RNeasy Mini kit (Qiagen). Quantity and quality of sgRNA were analyzed using a NanoDrop and agarose gel electrophoresis. sgRNA cleaving activity was confirmed by an in vivo assay.