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

CRISPR-Cas9がヒト細胞内のRNAで阻害されてしまう現象を発見し、iPS細胞での効率的な相同組み換えゲノム編集技術を実現. 京都大学プレスリリース. 2021-03-12.A simple step to enhance CRISPR-Cas9 genome editing. 京都大学プレスリリース. 2021-03-12.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

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

iPS細胞を使った遺伝子修復に成功 --デュシェンヌ型筋ジストロフィーの変異遺伝子を修復--. 京都大学プレスリリース. 2014-11-27.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

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

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

Phenotypic correction of hemophilia A mice by injection of <i>piggyBac</i> vectors.

<p>(A) Bleeding time of both hemophilia A mice (n = 6) and hemophilia A mice treated with <i>piggyBac</i> vector expressing full-length Factor VIII (n = 5) assessed by tail-clip assay. (B) Immunohistochemical analysis of liver tissue from non-treated hemophilia A mice and hemophilia A mice treated with <i>piggyBac</i> vector expressing full-length FVIII. Scale bar represents 50 µm (x400: original magnification). (C) Total RNA were extracted from the mouse liver treated with <i>piggyBac</i> vectors with 4 weeks interval, and quantified the level of transgene mRNA by qRT-PCR using human FVIII light-chain primers. Expression values were normalized to the level of <i>GAPDH</i> mRNA.</p

Delivery of Full-Length Factor VIII Using a <i>piggyBac</i> Transposon Vector to Correct a Mouse Model of Hemophilia A

<div><p>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 <i>piggyBac</i> 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 <i>piggyBac</i> 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.<i>piggyBac</i> transposon vectors can facilitate the long-term expression of therapeutic transgenes <i>in vitro</i> and <i>in vivo</i>. This novel gene transfer strategy should provide safe and efficient delivery of FVIII.</p></div

<i>piggyBac</i> vectors to express Factor VIII cDNAs.

<p>(A) Schematic diagram of <i>piggyBac</i> vectors expressing EGFP, B-domain–deleted human FVIII, and full-length human FVIII under the control of the human EF1α promoter. The PBaseII vector expresses <i>piggyBac</i> transposase under the control of the CAG promoter. IRES: internal ribosomal entry site. (B) Copy number of genomic <i>piggyBac</i> vectors. Indicated <i>piggyBac</i> vectors were transfected into 293T cells and selected with puromycin resistance. Approximately three months after transduction, genomic DNAs were extracted, and <i>piggyBac</i> vector copy numbers were assessed by real-time PCR using the <i>piggyBac</i> 5′ TR primers. The data are normalized to a haploid genome calculated from the copy number of NANOG gene. *: <i>P</i><0.05 by two-sided Student's <i>t</i> test (n = 3). N.S.: Not significant. (C) The sizes of inserted FVIII cDNA (2,219 bp for BDD and 4,901 bp for full-length FVIII) were confirmed by genomic PCR using the hF8insertC primers flanking the B-domain (indicated as small arrows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104957#pone-0104957-g001" target="_blank">Figure 1A</a>).</p

Full-length FVIII can be expressed at levels as high as BDD FVIII.

<p>(A, B) Total mRNAs were extracted from 293T cells (A) or human iPS cells (B), and the level of expression was assessed by quantitative RT-PCR. Expression values were normalized to the level of <i>GAPDH</i> mRNA. (C, D) Secretion of functional FVIII measured by aPTT assay. Culture supernatants of transfected cells were harvested 3 days after medium change and subjected to aPTT assay to measure the coagulation activity of secreted FVIII protein in 293T cells (C) or human iPS cells (D). Recombinant FVIII product was used to generate a standard curve. Normal FVIII activity (100%) represents 1 U/ml ( = 1000 mU/ml). *: <i>P</i><0.05 by two-sided Student's <i>t</i> test (n = 3).</p