Optimized electroporation of CRISPR-Cas9/gRNA ribonucleoprotein complex for selection-free homologous recombination in human pluripotent stem cells

Selection-free, scarless genome editing in human pluripotent stem cells (PSCs) by utilizing ribonucleoprotein (RNP) of CRISPR-Cas9 is a useful tool for a variety of applications. However, the process can be hampered by time-consuming subcloning steps and inefficient delivery of the RNP complex and ssDNA template. Here, we describe the optimized protocol to introduce a single nucleotide change or a loxP site insertion in feeder-free, xeno-free iPSCs by utilizing MaxCyte and 4D-Nucleofector electroporators. For complete details on the use and execution of this protocol, please refer to Kagita et al. (2021) and Xu et al. (2019)

Dual CRISPR-Cas3 system for inducing multi-exon skipping in DMD patient-derived iPSCs

DMD患者さん由来iPS細胞で複数のエクソンスキッピングを誘導するデュアルCRISPR-Cas3システムの開発. 京都大学プレスリリース. 2023-08-25.Exploring New Avenues in DMD Treatment: CRISPR-Cas3's Multi-Exon Skipping Approach. 京都大学プレスリリース. 2023-08-28.To restore dystrophin protein in various mutation patterns of Duchenne muscular dystrophy (DMD), the multi-exon skipping (MES) approach has been investigated. However, only limited techniques are available to induce a large deletion to cover the target exons spread over several hundred kilobases. Here, we utilized the CRISPR-Cas3 system for MES induction and showed that dual crRNAs could induce a large deletion at the dystrophin exon 45–55 region (∼340 kb), which can be applied to various types of DMD patients. We developed a two-color SSA-based reporter system for Cas3 to enrich the genome-edited cell population and demonstrated that MES induction restored dystrophin protein in DMD-iPSCs with three distinct mutations. Whole-genome sequencing and distance analysis detected no significant off-target deletion near the putative crRNA binding sites. Altogether, dual CRISPR-Cas3 is a promising tool to induce a gigantic genomic deletion and restore dystrophin protein via MES induction