Efficient ssODN-Mediated Targeting by Avoiding Cellular Inhibitory RNAs through Precomplexed CRISPR-Cas9/sgRNA Ribonucleoprotein.

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.

Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping.

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.

CRISPR-Cas3 induces broad and unidirectional genome editing in human cells.

Although single-component Class 2 CRISPR systems, such as type II Cas9 or type V Cas12a (Cpf1), are widely used for genome editing in eukaryotic cells, the application of multi-component Class 1 CRISPR has been less developed. Here we demonstrate that type I-E CRISPR mediates distinct DNA cleavage activity in human cells. Notably, Cas3, which possesses helicase and nuclease activity, predominantly triggered several thousand base pair deletions upstream of the 5'-ARG protospacer adjacent motif (PAM), without prominent off-target activity. This Cas3-mediated directional and broad DNA degradation can be used to introduce functional gene knockouts and knock-ins. As an example of potential therapeutic applications, we show Cas3-mediated exon-skipping of the Duchenne muscular dystrophy (DMD) gene in patient-induced pluripotent stem cells (iPSCs). These findings broaden our understanding of the Class 1 CRISPR system, which may serve as a unique genome editing tool in eukaryotic cells distinct from the Class 2 CRISPR system.

Targeted Disruption of HLA Genes via CRISPR-Cas9 Generates iPSCs with Enhanced Immune Compatibility.

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.

Efficient mRNA delivery system utilizing chimeric VSVG-L7Ae virus-like particles.

The delivery of mRNA is advantageous over DNA delivery as it is transient and does not carry the risk of genomic DNA integration. However, there are currently few efficient mRNA delivery options available, especially for hard-to-transfect cell types, and thus new delivery methods are needed. To this end, we have established a novel mRNA delivery system utilizing chimeric virus-like particles (VLPs). We generated a novel VLP by fusing protein G of Vesicular stomatitis virus (VSV-G) with a ribosomal protein L7Ae of Archeoglobus fulgidus. This system allowed the efficient delivery of EGFP mRNA which was independent from the presence of BoxC/D motif in the mRNA sequence. Our VSVG-L7Ae VLP system demonstrated high transduction efficacy in hard-to-transfect cell lines, such as human induced pluripotent stem cells (iPS cells) and monocytes. In summary, this platform may serve as an efficient and transient transgene delivery tool for an mRNA of interest.

Site-specific randomization of the endogenous genome by a regulatable CRISPR-Cas9 piggyBac system in human cells.

Randomized mutagenesis at an endogenous chromosomal locus is a promising approach for protein engineering, functional assessment of regulatory elements, and modeling genetic variations. In mammalian cells, however, it is challenging to perform site-specific single-nucleotide substitution with single-stranded oligodeoxynucleotide (ssODN) donor templates due to insufficient homologous recombination and the infeasibility of positive selection. Here, we developed a DNA transposon based CRISPR-Cas9 regulated transcription and nuclear shuttling (CRONUS) system that enables the stable transduction of CRISPR-Cas9/sgRNA in broad cell types, but avoids undesired genome cleavage in the absence two chemical inducing molecules. Highly efficient single nucleotide alterations induced randomization of desired codons (up to 4 codons) at a defined genomic locus in various human cell lines, including human iPS cells. Thus, CRONUS provides a novel platform for modeling diseases and genetic variations.

Precise correction of the dystrophin gene in duchenne muscular dystrophy patient induced pluripotent stem cells by TALEN and CRISPR-Cas9.

Duchenne muscular dystrophy (DMD) is a severe muscle-degenerative disease caused by a mutation in the dystrophin gene. Genetic correction of patient-derived induced pluripotent stem cells (iPSCs) by TALENs or CRISPR-Cas9 holds promise for DMD gene therapy; however, the safety of such nuclease treatment must be determined. Using a unique k-mer database, we systematically identified a unique target region that reduces off-target sites. To restore the dystrophin protein, we performed three correction methods (exon skipping, frameshifting, and exon knockin) in DMD-patient-derived iPSCs, and found that exon knockin was the most effective approach. We further investigated the genomic integrity by karyotyping, copy number variation array, and exome sequencing to identify clones with a minimal mutation load. Finally, we differentiated the corrected iPSCs toward skeletal muscle cells and successfully detected the expression of full-length dystrophin protein. These results provide an important framework for developing iPSC-based gene therapy for genetic disorders using programmable nucleases.

Delivery of full-length factor VIII using a piggyBac transposon vector to correct a mouse model of hemophilia A.

Viral vectors have been used for hemophilia A gene therapy. However, due to its large size, full-length Factor VIII (FVIII) cDNA has not been successfully delivered using conventional viral vectors. Moreover, viral vectors may pose safety risks, e.g., adverse immunological reactions or virus-mediated cytotoxicity. Here, we took advantages of the non-viral vector gene delivery system based on piggyBac DNA transposon to transfer the full-length FVIII cDNA, for the purpose of treating hemophilia A. We tested the efficiency of this new vector system in human 293T cells and iPS cells, and confirmed the expression of the full-length FVIII in culture media using activity-sensitive coagulation assays. Hydrodynamic injection of the piggyBac vectors into hemophilia A mice temporally treated with an immunosuppressant resulted in stable production of circulating FVIII for over 300 days without development of anti-FVIII antibodies. Furthermore, tail-clip assay revealed significant improvement of blood coagulation time in the treated mice. piggyBac transposon vectors can facilitate the long-term expression of therapeutic transgenes in vitro and in vivo. This novel gene transfer strategy should provide safe and efficient delivery of FVIII.

Double bromodomain protein BET-1 and MYST HATs establish and maintain stable cell fates in C. elegans.

The maintenance of cell fate is important for normal development and tissue homeostasis. Epigenetic mechanisms, including histone modifications, are likely to play crucial roles in cell-fate maintenance. However, in contrast to the established functions of histone methylation, which are mediated by the polycomb proteins, the roles of histone acetylation in cell-fate maintenance are poorly understood. Here, we show that the C. elegans acetylated-histone-binding protein BET-1 is required for the establishment and maintenance of stable fate in various lineages. In most bet-1 mutants, cells adopted the correct fate initially, but at later stages they often transformed into a different cell type. By expressing BET-1 at various times in development and examining the rescue of the Bet-1 phenotype, we showed that BET-1 functions both at the time of fate acquisition, to establish a stable fate, and at later stages, to maintain the established fate. Furthermore, the disruption of the MYST HATs perturbed the subnuclear localization of BET-1 and caused bet-1-like phenotypes, suggesting that BET-1 is recruited to its targets through acetylated histones. Our results therefore indicate that histone acetylation plays a crucial role in cell-fate maintenance.

Accumulation of FFA-1, the Xenopus homolog of Werner helicase, and DNA polymerase delta on chromatin in response to replication fork arrest.

Werner syndrome is a genetic disorder characterized by premature aging and cancer-prone symptoms, and is caused by mutation of the WRN gene. WRN is a member of the RecQ helicase family and is thought to function in processes implicated in DNA replication and repair to maintain genome stability; however, its precise function is still unclear. We found that replication fork arrest markedly enhances chromatin binding of focus-forming activity 1 (FFA-1), a Xenopus WRN homolog, in Xenopus egg extracts. In addition to FFA-1, DNA polymerase delta (Poldelta) and replication protein A, but not DNA polymerase epsilon and proliferating cell nuclear antigen, accumulated increasingly on replication-arrested chromatin. Elevated accumulation of these proteins was dependent on formation of pre-replicative complexes (pre-RCs). Double-strand break (DSB) formation also enhanced chromatin binding of FFA-1, but not Poldelta, independently of pre-RC formation. In contrast to FFA-1, chromatin binding of Xenopus Bloom syndrome helicase (xBLM) only slightly increased after replication arrest or DSB formation. Thus, WRN-specific, distinct processes can be reproduced in the in vitro system in egg extracts, and this system is useful for biochemical analysis of WRN functions during DNA metabolism.