Prolonged expression of the CRISPR-Cas9 nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. Thus, a transient delivery system is needed for therapeutic genome ...editing applications. Here, we develop an extracellular nanovesicle-based ribonucleoprotein delivery system named NanoMEDIC by utilizing two distinct homing mechanisms. Chemical induced dimerization recruits Cas9 protein into extracellular nanovesicles, and then a viral RNA packaging signal and two self-cleaving riboswitches tether and release sgRNA into nanovesicles. We demonstrate efficient genome editing in various hard-to-transfect cell types, including human induced pluripotent stem (iPS) cells, neurons, and myoblasts. NanoMEDIC also achieves over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy (DMD) patient iPS cells. Finally, single intramuscular injection of NanoMEDIC induces permanent genomic exon skipping in a luciferase reporter mouse and in mdx mice, indicating its utility for in vivo genome editing therapy of DMD and beyond.
Myotonic dystrophy type 1 (DM1) is caused by expanded CTG repeats (CTGexp) in the dystrophia myotonica protein kinase (DMPK) gene, and the transcription products, expanded CUG repeats, sequester ...muscleblind like splicing regulator 1 (MBNL1), resulting in the nuclear MBNL1 aggregation in the DM1 cells. Loss of MBNL1 function is the pivotal mechanism underlying the pathogenesis of DM1. To develop therapeutics for DM1, proper human in vitro models based on the pathologic mechanism of DM1 are required. In this study, we established robust in vitro skeletal muscle cell models of DM1 with patient-derived induced pluripotent stem cells (iPSCs) using the MyoD1-induced system and iPSCs-derived muscle stem cell (iMuSC) differentiation system. Our newly established DM1 models enable simple quantitative evaluation of nuclear MBNL1 aggregation and the downstream splicing defects. Quantitative analyses using the MyoD1-induced myotubes showed that CTGexp-deleted DM1 skeletal myotubes exhibited a reversal of MBNL1-related pathologies, and antisense oligonucleotide treatment recovered these disease phenotypes in the DM1-iPSCs-derived myotubes. Furthermore, iMuSC-derived myotubes exhibited higher maturity than the MyoD1-induced myotubes, which enabled us to recapitulate the SERCA1 splicing defect in the DM1-iMuSC-derived myotubes. Our quantitative and reproducible in vitro models for DM1 established using human iPSCs are promising for drug discovery against DM1.
•An FSHD2 patient-derived iPSC line with SMCHD1 mutation was generated.•The gene-corrected iPSC clone was also generated.•These clones are transgene-free and maintain normal karyotypes.
...Facioscapulohumeral muscular dystrophy type2 (FSHD2), which constitutes approximately 5% of total FSHD cases and develops the same symptoms as FSHD type 1 (FSHD1), is caused by various mutations in genes including SMCHD1. We report the generation and characterization of an iPSC line derived from an FSHD2 patient carrying the SMCHD1 p.Lys607Ter mutation and its gene-corrected iPSC line which are free from transgene. These iPSC lines maintained normal karyotype, presented typical morphology, expressed endogenous pluripotency markers, and could be differentiated into ectodermal, mesodermal and endodermal cells, confirming their pluripotency.
Induced pluripotent stem cells (iPSCs) have strong potential in regenerative medicine applications; however, immune rejection caused by HLA mismatching is a concern. B2M gene knockout and ...HLA-homozygous iPSC stocks can address this issue, but the former approach may induce NK cell activity and fail to present antigens, and it is challenging to recruit rare donors for the latter method. Here, we show two genome-editing strategies for making immunocompatible donor iPSCs. First, we generated HLA pseudo-homozygous iPSCs with allele-specific editing of HLA heterozygous iPSCs. Second, we generated HLA-C-retained iPSCs by disrupting both HLA-A and -B alleles to suppress the NK cell response while maintaining antigen presentation. HLA-C-retained iPSCs could evade T cells and NK cells in vitro and in vivo. We estimated that 12 lines of HLA-C-retained iPSCs combined with HLA-class II knockout are immunologically compatible with >90% of the world’s population, greatly facilitating iPSC-based regenerative medicine applications.
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•gRNA database, IFN-γ treatment, and HLA staining facilitated HLA editing in iPSCs•Allele-specific knockout of HLA genes generates pseudo-homozygous HLAs•Disrupting HLA-A/B, but retaining HLA-C, could evade CD8+ T and NK cell activities•Twelve lines of the HLA-C-retained cell would cover most of the HLA haplotypes
Xu et al. developed tailored HLA-editing strategies for facilitating the immunocompatibility of iPSCs. Selective deletion of HLA genes by CRISPR-Cas9 could evade T and NK cell activities by retaining HLA-C. To cover >90% of the world’s population, it is estimated that 12 lines of the HLA-C-retained iPSCs would be sufficient.
Combined with CRISPR-Cas9 technology and single-stranded oligodeoxynucleotides (ssODNs), specific single-nucleotide alterations can be introduced into a targeted genomic locus in induced pluripotent ...stem cells (iPSCs); however, ssODN knockin frequency is low compared with deletion induction. Although several Cas9 transduction methods have been reported, the biochemical behavior of CRISPR-Cas9 nuclease in mammalian cells is yet to be explored. Here, we investigated intrinsic cellular factors that affect Cas9 cleavage activity in vitro. We found that intracellular RNA, but not DNA or protein fractions, inhibits Cas9 from binding to single guide RNA (sgRNA) and reduces the enzymatic activity. To prevent this, precomplexing Cas9 and sgRNA before delivery into cells can lead to higher genome editing activity compared with Cas9 overexpression approaches. By optimizing electroporation parameters of precomplexed ribonucleoprotein and ssODN, we achieved efficiencies of single-nucleotide correction as high as 70% and loxP insertion up to 40%. Finally, we could replace the HLA-C1 allele with the C2 allele to generate histocompatibility leukocyte antigen custom-edited iPSCs.
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•Intracellular RNA, but not DNA or protein, inhibits Cas9 from binding to sgRNA•Precomplexing Cas9 and sgRNA is preferable to Cas9 overexpression approaches•Optimized electroporation permits high rates of ssODN-donor genome editing in iPSCs•loxP insertions and biallelic single-nucleotide corrections without antibiotics
Hotta et al. found that intracellular RNA, but not DNA or protein fractions, inhibits Cas9 from binding to sgRNA and reduces the enzymatic activity. To avoid this, precomplexing Cas9 and sgRNA could lead to higher genome editing activity than Cas9 overexpression. By optimizing electroporation conditions, biallelic single-nucleotide alterations and loxP insertion were feasible without antibiotic selection in iPSCs.
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